Polypeptides having endoglucanase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 12/293,735, filed Mar. 10, 2009, which is a National Phase filingunder 35 U.S.C. §371 of International Application No. PCT/US07/63710,filed Mar. 9, 2007, which claims priority to U.S. ProvisionalApplication Ser. No. 60/784,088, filed Mar. 20, 2006. The contents ofthese applications are hereby incorporated by reference in theirentireties.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to deposits of biological materialwhich have been made at the Northern Regional Research Center (NRRL)under the Budapest Treaty and assigned accession numbers NRRL B-30900N,NRRL B-30902, NRRL B-30903, and NRRL B-30904, which microbial depositsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingendoglucanase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiohydrolase Iis a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) activity whichcatalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellotetriose, or any beta-1,4-linked glucose containing polymer,releasing cellobiose from the reducing ends of the chain.Cellobiohydrolase II is a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91)activity which catalyzes the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, cellotetriose, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the non-reducing ends ofthe chain. Cellobiose is a water-soluble beta-1,4-linked dimer ofglucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

Roy et al., 1990, Journal of General Microbiology 136: 1967-1972,disclose the purification and properties of an extracellularendoglucanase from Myceliophthora thermophila ATCC 48104. Chemoglazov etal., 1988, Biokhimiya 53: 475-482, disclose the isolation, purification,and substrate specificity of an endoglucanase from Myceliophthorathermophila. Klyosov et al., 1988, Biotechnology Letters 10: 351-354,disclose a thermostable endoglucanase from Myceliophthora thermophila.Guzhova and Loginova, 1987, Prikladnaya Biokhimiya I Mikrobiologiya 23:820-825, disclose cellulolytic enzymes from Myceliophthora thermophila.Rabinovich et al., 1986, Bioorganicheskaya Khimiya 12: 1549-1560,disclose the purification and characterization of an endoglucanase fromMyceliophthora thermophila. Svistova et al., 1986, Mikrobiologiya 55:49-54, disclose the regulation of cellulose biosynthesis inMyceliophthora thermophila. Bhat and Maheshwari, 1987, Applied andEnvironmental Microbiology 53: 2175-2182, disclose the activity ofcomponents of the extracellular cellulose system of Myceliophthorathermophila. Klyosov et al., 1987, Prikladnaya Biokhimiya IMikrobiologiya 23: 44-50, disclose a thermostable endoglucanase fromMyceliophthora thermophila. Jorgensen et al., 2003, Enzyme and MicrobialTechnology 32: 851-861, and Thygesen et al., 2003, Enzyme and MicrobialTechnology 32: 606-615, disclose cellulose-degrading enzymes fromPenicillium brasilianum IBT 20888.

It would be an advantage in the art to identify new endoglucanaseshaving improved properties, such as improved hydrolysis rate, betterthermal stability, reduced adsorption to lignin, and ability tohydrolyze non-cellulosic components of biomass, such as hemicellulose,in addition to hydrolyzing cellulose. Endoglucanases with a broad rangeof side activities on hemicellulose can be especially beneficial forimproving the overall hydrolysis yield of complex, hemicellulose-richbiomass substrates.

It is an object of the present invention to provide improvedpolypeptides having endoglucanase activity and polynucleotides encodingthe polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingendoglucanase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence which has at least80% identity with the mature polypeptide of SEQ ID NO: 4 or SEQ ID NO:10, at least 85% identity with the mature polypeptide of SEQ ID NO: 6,or at least 75% identity with the mature polypeptide of SEQ ID NO: 8;

(b) a polypeptide which is encoded by a polynucleotide which hybridizesunder at least high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 3 or SEQ ID NO: 9 or thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) a full-length complementarystrand of (i) or (ii);

(c) a polypeptide which is encoded by a polynucleotide having at least80% identity with the mature polypeptide coding sequence of SEQ ID NO: 3or SEQ ID NO: 9, at least 85% identity with the mature polypeptidecoding sequence of SEQ ID NO: 5, or at least 75% identity with themature polypeptide coding sequence of SEQ ID NO: 7; and

(d) a variant comprising a substitution, deletion, and/or insertion ofone or more amino acids of the mature polypeptide of SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

The present invention also relates to isolated polynucleotides encodingpolypeptides having endoglucanase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence which has at least 80% identity with the mature polypeptide ofSEQ ID NO: 4 or SEQ ID NO: 10, at least 85% identity with the maturepolypeptide of SEQ ID NO: 6, or at least 75% identity with the maturepolypeptide of SEQ ID NO: 8;

(b) a polynucleotide which hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 3 orSEQ ID NO: 9 or the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) acomplementary strand of (i) or (ii);

(c) a polynucleotide having at least 80% identity with the maturepolypeptide coding sequence of SEQ ID NO: 3 or SEQ ID NO: 9, at least85% identity with the mature polypeptide coding sequence of SEQ ID NO:5, or at least 75% identity with the mature polypeptide coding sequenceof SEQ ID NO: 7; and

(d) a polynucleotide encoding a variant comprising a substitution,deletion, and/or insertion of one or more amino acids of the maturepolypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10.

In a preferred aspect, the mature polypeptide is amino acids 17 to 389of SEQ ID NO: 4. In another preferred aspect, the mature polypeptide isamino acids 16 to 397 of SEQ ID NO: 6. In another preferred aspect, themature polypeptide is amino acids 22 to 429 of SEQ ID NO: 8. In anotherpreferred aspect, the mature polypeptide is amino acids 25 to 421 of SEQID NO: 10. In another preferred aspect, the mature polypeptide codingsequence is nucleotides 67 to 1185 of SEQ ID NO: 3. In another preferredaspect, the mature polypeptide coding sequence is nucleotides 84 to 1229of SEQ ID NO: 5. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 77 to 1300 of SEQ ID NO: 7. In anotherpreferred aspect, the mature polypeptide coding sequence is nucleotides73 to 1468 of SEQ ID NO: 9.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingendoglucanase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide in a cell, comprising administering to thecell or expressing in the cell a double-stranded RNA (dsRNA) molecule,wherein the dsRNA comprises a subsequence of a polynucleotide of thepresent invention. The present also relates to such a double-strandedinhibitory RNA (dsRNA) molecule, wherein optionally the dsRNA is ansiRNA or an miRNA molecule.

The present invention also relates to methods of using the polypeptideshaving endoglucanase activity in the conversion of cellulose to glucoseand various substances.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding such a polypeptide having endoglucanaseactivity.

The present invention also relates to methods for producing such apolypeptide having endoglucanase activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingsuch a polypeptide having endoglucanase activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to a nucleotide sequence encoding a signal peptide comprising orconsisting of amino acids 1 to 16 of SEQ ID NO: 4, amino acids 1 to 15of SEQ ID NO: 6, amino acids 1 to 21 of SEQ ID NO: 8, or amino acids 1to 16 of SEQ ID NO: 10 and a second nucleotide sequence encoding apropeptide comprising or consisting of amino acids 17 to 24 of SEQ IDNO: 10, wherein the gene is foreign to the first and second nucleotidesequences

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pCIC161.

FIG. 2 shows the genomic DNA sequence and the deduced amino acidsequence of a Myceliophthora thermophila CBS 117.65 endoglucanase (SEQID NOs: 3 and 4, respectively).

FIG. 3 shows a restriction map of pA2C161.

FIG. 4 shows a restriction map of pCIC453.

FIG. 5 shows a restriction map of pA2C453.

FIG. 6 shows the cDNA sequence and the deduced amino acid sequence of abasidiomycete CBS 495.95 endoglucanase (SEQ ID NOs: 5 and 6,respectively).

FIG. 7 shows a restriction map of pCIC486.

FIG. 8 shows a restriction map of pA2C486.

FIG. 9 shows the cDNA sequence and the deduced amino acid sequence of abasidiomycete CBS 494.95 endoglucanase (SEQ ID NOs: 7 and 8,respectively).

FIGS. 10A and 10B show the genomic DNA sequence and the deduced aminoacid sequence of a Penicillium brasilianum strain IBT 20888endoglucanase (SEQ ID NOs: 9 and 10, respectively).

FIG. 11 shows a restriction map of pKBK03.

FIG. 12 shows a restriction map of pPBCel5C.

FIG. 13 shows the specific activity of the Penicillium brasilianum IBT20888 CEL5C endoglucanase at different pH values and 50° C. (n=2).

FIG. 14 shows the specific activity of the Penicillium brasilianum IBT20888 CEL5C endoglucanase at different temperatures and pH 4.8 (n=2).

FIG. 15 shows the residual activity of the Penicillium brasilianum IBT20888 CEL5C endoglucanase after 20 hours of incubation at different pHvalues and 25° C. and 50° C. (n=2).

FIG. 16 shows the relative activity on PASC (2 mg/ml) as a function oftemperature for basidiomycete CBS 494.95 and basidiomycete CBS 495.95 atpH 5.0.

FIG. 17 shows the relative conversion of PASC (2 mg/ml) as a function oftemperature after 45 hours of hydrolysis with basidiomycete CBS 494.95and basidiomycete CBS 495.95 (0.5 mg protein per g of PASC) at pH 5.0.

FIG. 18 shows a comparison of endoglucanases from Myceliophthorathermophila CBS 117.65, basidiomycete CBS 494.95, basidiomycete CBS495.95, and Trichoderma reesei for production of reducing sugars frombeta-glucan (1% w/v) after 2-hour hydrolysis reaction at pH 5.5 and 60°C.

FIG. 19 shows a comparison of endoglucanases from Myceliophthorathermophila CBS 117.65, basidiomycete CBS 494.95, basidiomycete CBS495.95, and Trichoderma reesei for production of reducing sugars frombeta-glucan (1% w/v) after 2-hour hydrolysis reaction at pH 5.5 and 60°C.

DEFINITIONS

Endoglucanase activity: The term “endoglucanase activity” is definedherein as an endo-1,4-beta-D-glucan 4-glucanohydrolase (E.C. No.3.2.1.4) which catalyses the endohydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and otherplant material containing cellulosic components. For purposes of thepresent invention, endoglucanase activity is determined usingcarboxymethyl cellulose (CMC) hydrolysis according to the procedure ofGhose, 1987, Pure and Appl. Chem. 59: 257-268. One unit of endoglucanaseactivity is defined as 1.0 μmole of reducing sugars produced per minuteat 50° C., pH 4.8.

In a preferred aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward one or moresubstrates selected from the group consisting of xylan, xyloglucan,arabinoxylan, 1,4-beta-D-mannan, and galactomannan. The activity of thepolypeptides having endoglucanase activity on these polysaccharidesubstrates is determined as percent of the substrate hydrolyzed toreducing sugars after incubating the substrate (5 mg per ml) with apolypeptide having endoglucanase activity of the present invention (5 mgprotein per g of substrate) for 24 hours with intermittent stirring atpH 5.0 (50 mM sodium acetate) and 50° C. Reducing sugars in hydrolysismixtures are determined by the p-hydroxybenzoic acid hydrazide (PHBAH)assay.

In a more preferred aspect, the polypeptides of the present inventionhaving endoglucanase activity further have enzyme activity toward xylan.In another more preferred aspect, the polypeptides of the presentinvention having endoglucanase activity further have enzyme activitytoward xyloglucan. In another more preferred aspect, the polypeptides ofthe present invention having endoglucanase activity further have enzymeactivity toward arabinoxylan. In another more preferred aspect, thepolypeptides of the present invention having endoglucanase activityfurther have enzyme activity toward 1,4-beta-D-mannan. In another morepreferred aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity towardgalactomannan. In another more preferred aspect, the polypeptides of thepresent invention having endoglucanase activity further have enzymeactivity toward xylan, xyloglucan, arabinoxylan, 1,4-beta-D-mannan,and/or galactomannan.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the endoglucanase activity of maturepolypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO:10.

Family 5 glycoside hydrolase or Family GH5: The term “Family 5 glycosidehydrolase” or “Family GH5” is defined herein as a polypeptide fallinginto the glycoside hydrolase Family 5 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively or recombinantly associated. This can be accomplished,for example, by preparing the polypeptide by means of well-knownrecombinant methods or by classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having endoglucanase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having endoglucanase activity.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of EMBOSS with gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 matrix. The output ofNeedle labeled “longest identity” is used as the percent identity and iscalculated as follows:(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of EMBOSS with gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL matrix. The output ofNeedle labeled “longest identity” is used as the percent identity and iscalculated as follows:(Identical Residues×100)/(Length of Alignment−Number of Gaps inAlignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein which gives an E value (or expectancy score) of lessthan 0.001 in a fasta search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the mature endoglucanase of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, or SEQ ID NO: 10.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; or a homologous sequencethereof; wherein the fragment has endoglucanase activity. In a preferredaspect, a fragment contains at least 295 amino acid residues, morepreferably at least 315 amino acid residues, and most preferably atleast 335 amino acid residues of the mature polypeptide of SEQ ID NO: 4or a homologous sequence thereof. In another preferred aspect, afragment contains at least 320 amino acid residues, more preferably atleast 340 amino acid residues, and most preferably at least 360 aminoacid residues of the mature polypeptide of SEQ ID NO: 6 or a homologoussequence thereof. In another preferred aspect, a fragment contains atleast 325 amino acid residues, more preferably at least 345 amino acidresidues, and most preferably at least 365 amino acid residues of themature polypeptide of SEQ ID NO: 8 or a homologous sequence thereof. Inanother preferred aspect, a fragment contains at least 335 amino acidresidues, more preferably at least 355 amino acid residues, and mostpreferably at least 375 amino acid residues of the mature polypeptide ofSEQ ID NO: 10 or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of the mature polypeptide coding sequence of SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; or a homologous sequence thereof;wherein the subsequence encodes a polypeptide fragment havingendoglucanase activity. In a preferred aspect, a subsequence contains atleast 885 nucleotides, more preferably at least 945 nucleotides, andmost preferably at least 1005 nucleotides of the mature polypeptidecoding sequence of SEQ ID NO: 3 or a homologous sequence thereof. Inanother preferred aspect, a subsequence contains at least 960nucleotides, more preferably at least 1020 nucleotides, and mostpreferably at least 1080 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 5 or a homologous sequence thereof. In anotherpreferred aspect, a subsequence contains at least 975 nucleotides, morepreferably at least 1035 nucleotides, and most preferably at least 1095nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 7 ora homologous sequence thereof. In another preferred aspect, asubsequence contains at least 1005 nucleotides, more preferably at least1065 nucleotides, and most preferably at least 1125 nucleotides of themature polypeptide coding sequence of SEQ ID NO: 9 or a homologoussequence thereof.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form. In particular, it is preferredthat the polynucleotides disclosed herein are in “essentially pureform”, i.e., that the polynucleotide preparation is essentially free ofother polynucleotide material with which it is natively or recombinantlyassociated. Herein, the term “substantially pure polynucleotide” issynonymous with the terms “isolated polynucleotide” and “polynucleotidein isolated form.” The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, or recombinant nucleotide sequence.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having endoglucanase activity.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10; or ahomologous sequence thereof; as well as genetic manipulation of the DNAencoding such a polypeptide. The modification can be substitutions,deletions and/or insertions of one or more amino acids as well asreplacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having endoglucanase activity produced by anorganism expressing a modified nucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9; or a homologous sequence thereof. The modifiednucleotide sequence is obtained through human intervention bymodification of the nucleotide sequence disclosed in SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; or a homologous sequencethereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Endoglucanase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10, of at least 60%, preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, 97%, 98%, or 99%, which have endoglucanase activity(hereinafter “homologous polypeptides”). In a preferred aspect, thehomologous polypeptides have an amino acid sequence which differs by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from themature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQID NO: 10.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 4. In another preferred aspect, a polypeptide comprisesamino acids 17 to 389 of SEQ ID NO: 4, or an allelic variant thereof; ora fragment thereof that has endoglucanase activity. In another preferredaspect, a polypeptide comprises amino acids 17 to 389 of SEQ ID NO: 4.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragmentthereof that has endoglucanase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, a polypeptideconsists of amino acids 17 to 389 of SEQ ID NO: 4 or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Inanother preferred aspect, a polypeptide consists of amino acids 17 to389 of SEQ ID NO: 4.

A polypeptide of the present invention preferably also comprises theamino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 6. In another preferred aspect, a polypeptide comprisesamino acids 16 to 397 of SEQ ID NO: 6, or an allelic variant thereof; ora fragment thereof that has endoglucanase activity. In another preferredaspect, a polypeptide comprises amino acids 16 to 397 of SEQ ID NO: 6.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragmentthereof that has endoglucanase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, a polypeptideconsists of amino acids 16 to 397 of SEQ ID NO: 6 or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Inanother preferred aspect, a polypeptide consists of amino acids 16 to397 of SEQ ID NO: 6.

A polypeptide of the present invention preferably also comprises theamino acid sequence of SEQ ID NO: 8 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 8. In another preferred aspect, a polypeptide comprisesamino acids 22 to 429 of SEQ ID NO: 8, or an allelic variant thereof; ora fragment thereof that has endoglucanase activity. In another preferredaspect, a polypeptide comprises amino acids 22 to 429 of SEQ ID NO: 8.In another preferred aspect, a polypeptide consists of the amino acidsequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragmentthereof that has endoglucanase activity. In another preferred aspect, apolypeptide consists of the amino acid sequence of SEQ ID NO: 8. Inanother preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 8. In another preferred aspect, a polypeptideconsists of amino acids 22 to 429 of SEQ ID NO: 8 or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Inanother preferred aspect, a polypeptide consists of amino acids 22 to429 of SEQ ID NO: 8.

A polypeptide of the present invention preferably also comprises theamino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In a preferred aspect,a polypeptide comprises the amino acid sequence of SEQ ID NO: 10. Inanother preferred aspect, a polypeptide comprises the mature polypeptideof SEQ ID NO: 10. In another preferred aspect, a polypeptide comprisesamino acids 25 to 421 of SEQ ID NO: 10, or an allelic variant thereof;or a fragment thereof that has endoglucanase activity. In anotherpreferred aspect, a polypeptide comprises amino acids 25 to 421 of SEQID NO: 10. In another preferred aspect, a polypeptide consists of theamino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or afragment thereof that has endoglucanase activity. In another preferredaspect, a polypeptide consists of the amino acid sequence of SEQ ID NO:10. In another preferred aspect, a polypeptide consists of the maturepolypeptide of SEQ ID NO: 10. In another preferred aspect, a polypeptideconsists of amino acids 25 to 421 of SEQ ID NO: 10 or an allelic variantthereof; or a fragment thereof that has endoglucanase activity. Inanother preferred aspect, a polypeptide consists of amino acids 25 to421 of SEQ ID NO: 10.

In a second aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity which are encoded bypolynucleotides which hybridize under very low stringency conditions,preferably low stringency conditions, more preferably medium stringencyconditions, more preferably medium-high stringency conditions, even morepreferably high stringency conditions, and most preferably very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 3 or SEQ ID NO: 9 or the genomic DNA sequence comprising the maturepolypeptide coding sequence of SEQ ID NO: 5 or SEQ ID NO: 7, (iii) asubsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii),or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Asubsequence of the mature polypeptide coding sequence of SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has endoglucanase activity. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 67 to 1185 of SEQ ID NO: 3.In another preferred aspect, the mature polypeptide coding sequence isnucleotides 84 to 1229 of SEQ ID NO: 5. In another preferred aspect, themature polypeptide coding sequence is nucleotides 77 to 1300 of SEQ IDNO: 7. In another preferred aspect, the mature polypeptide codingsequence is nucleotides 73 to 1468 of SEQ ID NO: 9. In another preferredaspect, the complementary strand is the full-length complementary strandof the mature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5,SEQ ID NO: 7, or SEQ ID NO: 9.

The nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, orSEQ ID NO: 9; or a subsequence thereof; as well as the amino acidsequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10;or a fragment thereof; may be used to design a nucleic acid probe toidentify and clone DNA encoding polypeptides having endoglucanaseactivity from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, preferably at least 25, more preferably at least 35, and mostpreferably at least 70 nucleotides in length. It is, however, preferredthat the nucleic acid probe is at least 100 nucleotides in length. Forexample, the nucleic acid probe may be at least 200 nucleotides,preferably at least 300 nucleotides, more preferably at least 400nucleotides, or most preferably at least 500 nucleotides in length. Evenlonger probes may be used, e.g., nucleic acid probes which are at least600 nucleotides, at least preferably at least 700 nucleotides, morepreferably at least 800 nucleotides, or most preferably at least 900nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having endoglucanaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO:9; or a subsequence thereof; the carrier material is preferably used ina Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9; the cDNA sequence containedin the mature polypeptide coding sequence of SEQ ID NO: 3 or SEQ ID NO:9 or the genomic DNA sequence comprising the mature polypeptide codingsequence of SEQ ID NO: 5 or SEQ ID NO: 7; its complementary strand; or asubsequence thereof; under very low to very high stringency conditions.Molecules to which the nucleic acid probe hybridizes under theseconditions can be detected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 3. In another preferred aspect, thenucleic acid probe is nucleotides 67 to 1185 of SEQ ID NO: 3. In anotherpreferred aspect, the nucleic acid probe is a polynucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 4, or a subsequence thereof.In another preferred aspect, the nucleic acid probe is SEQ ID NO: 3. Inanother preferred aspect, the nucleic acid probe is the polynucleotidesequence contained in plasmid pCIC161 which is contained in E. coli NRRLB-30902, wherein the polynucleotide sequence thereof encodes apolypeptide having endoglucanase activity. In another preferred aspect,the nucleic acid probe is the mature polypeptide coding region containedin plasmid pCIC161 which is contained in E. coli NRRL B-30902.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is nucleotides 84 to 1229 of SEQ ID NO:5. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 6,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pCIC453 whichis contained in E. coli NRRL B-30903, wherein the polynucleotidesequence thereof encodes a polypeptide having endoglucanase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pCIC453 which iscontained in E. coli NRRL B-30903.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 7. In another preferredaspect, the nucleic acid probe is nucleotides 77 to 1300 of SEQ ID NO:7. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 8,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 7. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pCIC486 whichis contained in E. coli NRRL B-30904, wherein the polynucleotidesequence thereof encodes a polypeptide having endoglucanase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pCIC486 which iscontained in E. coli NRRL B-30904.

In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 9. In another preferredaspect, the nucleic acid probe is nucleotides 73 to 1468 of SEQ ID NO:9. In another preferred aspect, the nucleic acid probe is apolynucleotide sequence which encodes the polypeptide of SEQ ID NO: 10,or a subsequence thereof. In another preferred aspect, the nucleic acidprobe is SEQ ID NO: 9. In another preferred aspect, the nucleic acidprobe is the polynucleotide sequence contained in plasmid pPBCel5C whichis contained in E. coli NRRL B-30900N, wherein the polynucleotidesequence thereof encodes a polypeptide having endoglucanase activity. Inanother preferred aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pPBCel5C which iscontained in E. coli NRRL B-30900N.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6× SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides encoded by polynucleotides comprising or consisting ofnucleotide sequences which have a degree of identity to the maturepolypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9 of at least 60%, preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, even more preferably at least 95%, and most preferably at least 97%identity, which encode an active polypeptide. In a preferred aspect, themature polypeptide coding sequence is nucleotides 67 to 1185 of SEQ IDNO: 3. In another preferred aspect, the mature polypeptide codingsequence is nucleotides 84 to 1229 of SEQ ID NO: 5. In another preferredaspect, the mature polypeptide coding sequence is nucleotides 77 to 1300of SEQ ID NO: 7. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 73 to 1468 of SEQ ID NO: 9. Seepolynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising a substitution, deletion, and/or insertion of one or moreamino acids of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, or SEQ ID NO: 10; or a homologous sequence thereof.Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,endoglucanase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, or SEQ ID NO: 10, such as amino acids 17 to 389 of SEQ ID NO:2, amino acids 16 to 397 of SEQ ID NO: 6, amino acids 22 to 429 of SEQID NO: 8, or amino acids 25 to 421 of SEQ ID NO: 10, is 10, preferably9, more preferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

Sources of Polypeptides Having Endoglucanase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide having endoglucanase activity of the present invention maybe a bacterial polypeptide. For example, the polypeptide may be a grampositive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide having [enzyme]activity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingendoglucanase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having endoglucanase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having endoglucanaseactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingendoglucanase activity.

A polypeptide having endoglucanase activity of the present invention mayalso be a fungal polypeptide, and more preferably a yeast polypeptidesuch as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having endoglucanaseactivity; or more preferably a filamentous fungal polypeptide such as anAcremonium, Aspergillus, Aureobasidium, Chrysosporium, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, orTrichoderma polypeptide having endoglucanase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having endoglucanaseactivity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Thielavia achromatica,Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis,Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielaviaperuviana, Thielavia spededonium, Thielavia setosa, Thielaviasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride polypeptide having endoglucanase activity.

In another preferred aspect, the polypeptide is a Penicilliumbrasilianum, Penicillium camembertii, Penicillium capsulatum,Penicillium chrysogenum, Penicillium citreonigrum, Penicillium citrinum,Penicillium claviforme, Penicillium corylophilum, Penicillium crustosum,Penicillium digitatum, Penicillium expansum, Penicillium funiculosum,Penicillium glabrum, Penicillium granulatum, Penicillium griseofulvum,Penicillium islandicum, Penicillium italicum, Penicillium janthinellum,Penicillium lividum, Penicillium megasporum, Penicillium melinfi,Penicillium notatum, Penicillium oxalicum, Penicillium puberulum,Penicillium purpurescens, Penicillium purpurogenum, Penicilliumroquefortii, Penicillium rugulosum, Penicillium spinulosum, Penicilliumwaksmanii, or Penicillium sp. polypeptide having endoglucanase activity.

In a more preferred aspect, the polypeptide is a Myceliophthorathermophila polypeptide, and most preferably a Myceliophthorathermophila CBS 111.65 polypeptide, e.g., the polypeptide of SEQ ID NO:4, or the mature polypeptide thereof.

In another more preferred aspect, the polypeptide is a basidiomycete CBS495.95 polypeptide, e.g., the polypeptide of SEQ ID NO: 6, or the maturepolypeptide thereof.

In another more preferred aspect, the polypeptide is a basidiomycete CBS494.95 polypeptide, e.g., the polypeptide of SEQ ID NO: 8, or the maturepolypeptide thereof.

In another more preferred aspect, the polypeptide is a Penicilliumbrasilianum polypeptide, and most preferably a Penicillium brasilianumIBT 20888 polypeptide, e.g., the polypeptide of SEQ ID NO: 10, or themature polypeptide thereof.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having endoglucanase activity from the fusion protein.

Examples of cleavage sites include, but are not limited to, a Kex2 sitewhich encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to an isolated polynucleotidecomprising or consisting of a nucleotide sequence which encodes apolypeptide of the present invention having endoglucanase activity.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 3. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pCIC161 whichis contained in E. coli NRRL B-30902. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 3. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 67 to 1185 ofSEQ ID NO: 3. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding region containedin plasmid pCIC161 which is contained in E. coli NRRL B-30902. Thepresent invention also encompasses nucleotide sequences which encodepolypeptides comprising or consisting of the amino acid sequence of SEQID NO: 4 or the mature polypeptide thereof, which differ from SEQ ID NO:3 or the mature polypeptide coding sequence thereof by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 3 which encode fragments of SEQ ID NO: 4 thathave endoglucanase activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 5. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pCIC453 which is contained in E. coli NRRL B-30903. In anotherpreferred aspect, the nucleotide sequence comprises or consists of themature polypeptide coding region of SEQ ID NO: 5. In another preferredaspect, the nucleotide sequence comprises or consists of nucleotides 84to 1229 of SEQ ID NO: 5. In another more preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region contained in plasmid pCIC453 which is contained in E. coliNRRL B-30903. The present invention also encompasses nucleotidesequences which encode polypeptides comprising or consisting of theamino acid sequence of SEQ ID NO: 6 or the mature polypeptide thereof,which differ from SEQ ID NO: 5 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 5 which encodefragments of SEQ ID NO: 6 that have endoglucanase activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 7. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pCIC486 which is contained in E. coli NRRL B-30904. In anotherpreferred aspect, the nucleotide sequence comprises or consists of themature polypeptide coding region of SEQ ID NO: 7. In another preferredaspect, the nucleotide sequence comprises or consists of nucleotides 77to 1300 of SEQ ID NO: 7. In another more preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region contained in plasmid pCIC486 which is contained in E. coliNRRL B-30904. The present invention also encompasses nucleotidesequences which encode polypeptides comprising or consisting of theamino acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof,which differ from SEQ ID NO: 7 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 7 which encodefragments of SEQ ID NO: 8 that have endoglucanase activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 9. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pPBCel5C which is contained in E. coli NRRL B-30900N. In anotherpreferred aspect, the nucleotide sequence comprises or consists of themature polypeptide coding region of SEQ ID NO: 9. In another preferredaspect, the nucleotide sequence comprises or consists of nucleotides 73to 1468 of SEQ ID NO: 9. In another more preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region contained in plasmid pPBCel5C which is contained in E.coli NRRL B-30900N. The present invention also encompasses nucleotidesequences which encode polypeptides comprising or consisting of theamino acid sequence of SEQ ID NO: 10 or the mature polypeptide thereof,which differ from SEQ ID NO: 9 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 9 which encodefragments of SEQ ID NO: 10 that have endoglucanase activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9,in which the mutant nucleotide sequence encodes the mature polypeptideof SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10. In apreferred aspect, the mature polypeptide is amino acids 17 to 389 of SEQID NO: 4. In another preferred aspect, the mature polypeptide is aminoacids 16 to 397 of SEQ ID NO: 6. In another preferred aspect, the maturepolypeptide is amino acids 22 to 429 of SEQ ID NO: 8. In anotherpreferred aspect, the mature polypeptide is amino acids 25 to 421 of SEQID NO: 10.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Myceliophthora thermophila CBS 117.65,basidiomycete CBS 494.95, or basidiomycete CBS 495.95, or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences which have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9 of at least 60%, preferably atleast 65%, more preferably at least 70%, more preferably at least 75%,more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, even more preferably at least 95%, and mostpreferably at least 97% identity, which encode an active polypeptide. Ina preferred aspect, the mature polypeptide coding sequence isnucleotides 67 to 1185 of SEQ ID NO: 3. In another preferred aspect, themature polypeptide coding sequence is nucleotides 84 to 1229 of SEQ IDNO: 5. In another preferred aspect, the mature polypeptide codingsequence is nucleotides 77 to 1300 of SEQ ID NO: 7. In another preferredaspect, the mature polypeptide coding sequence is nucleotides 73 to 1468of SEQ ID NO: 9.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for endoglucanase activity toidentify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith etal., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,or SEQ ID NO: 9, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 3 or SEQ ID NO: 9 or thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) a complementary strand of (i)or (ii); or allelic variants and subsequences thereof (Sambrook et al.,1989, supra), as defined herein. In a preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 3 is nucleotides 67 to 1185.In another preferred aspect, the mature polypeptide coding sequence ofSEQ ID NO: 5 is nucleotides 84 to 1229. In another preferred aspect, themature polypeptide coding sequence of SEQ ID NO: 7 is nucleotides 77 to1300. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 9 is nucleotides 73 to 1768.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) themature polypeptide coding sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, or SEQ ID NO: 9, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 3 or SEQ ID NO: 9 or thegenomic DNA sequence comprising the mature polypeptide coding sequenceof SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) a complementary strand of (i)or (ii); and (b) isolating the hybridizing polynucleotide, which encodesa polypeptide having endoglucanase activity. In a preferred aspect, themature polypeptide coding sequence of SEQ ID NO: 3 is nucleotides 67 to1185. In another preferred aspect, the mature polypeptide codingsequence of SEQ ID NO: 5 is nucleotides 84 to 1229. In another preferredaspect, the mature polypeptide coding sequence of SEQ ID NO: 7 isnucleotides 77 to 1300. In another preferred aspect, the maturepolypeptide coding sequence of SEQ ID NO: 9 is nucleotides 73 to 1768.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dada (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 16 of SEQ ID NO: 4. In another preferred aspect, the signalpeptide coding region is nucleotides 19 to 69 of SEQ ID NO: 3.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 15 of SEQ ID NO: 6. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 39 to83 of SEQ ID NO: 5.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 21 of SEQ ID NO: 8. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 14 to76 of SEQ ID NO: 7.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 16 of SEQ ID NO: 10. In another preferred aspect, thesignal peptide coding region comprises or consists of nucleotides 1 to48 of SEQ ID NO: 9.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propeptide is generallyinactive and can be converted to a mature active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred aspect, the propeptide comprises or consists of aminoacids 17 to 24 of SEQ ID NO: 10. In another preferred aspect, thepropeptide coding region comprises or consists of nucleotides 49 to 72of SEQ ID NO: 9.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a polynucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMRβ1permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyceslividans.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98:6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium brasilianum,Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. In a preferred aspect,the cell is of the genus Myceliophthora. In a more preferred aspect, thecell is Myceliophthora thermophila. In a most preferred aspect, the cellis Myceliophthora thermophila CBS 117.65. In another preferred aspect,the cell is basidiomycete CBS 494.95. In another preferred aspect, thecell is basidiomycete CBS 495.95. In another preferred aspect, the cellis of the genus Penicillium. In another more preferred aspect, the cellis Penicillium brasilianum. In another most preferred aspect, the cellis Penicillium brasilianum IBT 20888.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, wherein the mutantnucleotide sequence encodes a polypeptide which comprises or consists ofthe mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, orSEQ ID NO: 10, and (b) recovering the polypeptide.

In a preferred aspect, the mature polypeptide of SEQ ID NO: 4 is aminoacids 17 to 389. In another preferred aspect, the mature polypeptide ofSEQ ID NO: 6 is amino acids 16 to 397. In another preferred aspect, themature polypeptide of SEQ ID NO: 8 is amino acids 22 to 429. In anotherpreferred aspect, the mature polypeptide of SEQ ID NO: 10 is amino acids25 to 421. In another preferred aspect, the mature polypeptide codingsequence is nucleotides 67 to 1185 of SEQ ID NO: 3. In another preferredaspect, the mature polypeptide coding sequence is nucleotides 84 to 1229of SEQ ID NO: 5. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 77 to 1300 of SEQ ID NO: 7. In anotherpreferred aspect, the mature polypeptide coding sequence is nucleotides73 to 1468 of SEQ ID NO: 9.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having endoglucanase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having endoglucanase activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Endoglucanase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of endoglucanase activityby fermentation of a cell which produces both a polypeptide of thepresent invention as well as the protein product of interest by addingan effective amount of an agent capable of inhibiting endoglucanaseactivity to the fermentation broth before, during, or after thefermentation has been completed, recovering the product of interest fromthe fermentation broth, and optionally subjecting the recovered productto further purification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of endoglucanase activityby cultivating the cell under conditions permitting the expression ofthe product, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the endoglucanase activitysubstantially, and recovering the product from the culture broth.Alternatively, the combined pH and temperature treatment may beperformed on an enzyme preparation recovered from the culture broth. Thecombined pH and temperature treatment may optionally be used incombination with a treatment with an endoglucanase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the endoglucanase activity. Complete removal of endoglucanaseactivity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-3 or 10-11 and a temperature in the range of atleast 75-85° C. for a sufficient period of time to attain the desiredeffect, where typically, 1 to 3 hours is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyendoglucanase-free product is of particular interest in the productionof eukaryotic polypeptides, in particular fungal proteins such asenzymes. The enzyme may be selected from, e.g., an amylolytic enzyme,lipolytic enzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase,or plant cell-wall degrading enzyme. Examples of such enzymes include anaminopeptidase, amylase, amyloglucosidase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Theendoglucanase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from endoglucanase activity which is produced by amethod of the present invention.

Methods of Inhibiting Expression of a Polypeptide

The present invention also relates to methods of inhibiting expressionof a polypeptide in a cell, comprising administering to the cell orexpressing in the cell a double-stranded RNA (dsRNA) molecule, whereinthe dsRNA comprises a subsequence or portion of a polynucleotide of thepresent invention. In a preferred aspect, the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. Inanother preferred aspect, the polypeptide has endoglucanase activity.

The dsRNA is preferably a small interfering RNA (sRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules for inhibiting expression of a polypeptide in a cell, whereinthe dsRNA comprises a subsequence or portion of a polynucleotideencoding the mature polypeptide of SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, or SEQ ID NO: 10. While the present invention is not limited byany particular mechanism of action, the dsRNA can enter a cell and causethe degradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencingtherapeutics. In one aspect, the invention provides methods toselectively degrade RNA using the dsRNAis of the present invention. Theprocess may be practiced in vitro, ex vivo or in vivo. In one aspect,the dsRNA molecules can be used to generate a loss-of-function mutationin a cell, an organ or an animal. Methods for making and using dsRNAmolecules to selectively degrade RNA are well known in the art, see, forexample, U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; and 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theendoglucanase activity of the composition has been increased, e.g., withan enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with a composition comprising an effective amount of apolypeptide having endoglucanase activity of the present invention. In apreferred aspect, the method further comprises recovering the degradedor converted cellulosic material.

The polypeptides and host cells of the present invention may be used inthe production of monosaccharides, disaccharides, and polysaccharides aschemical or fermentation feedstocks from cellulosic biomass for theproduction of ethanol, plastics, other products or intermediates. Thecomposition comprising the polypeptide having endoglucanase activity maybe in the form of a crude fermentation broth with or without the cellsremoved or in the form of a semi-purified or purified enzymepreparation. The composition can also comprise other proteins andenzymes useful in the processing of biomass, e.g., cellobiohydrolase,beta-glucosidase, hemicellulolytic enzymes, enhancers (WO 2005/074647,WO 2005/074656), etc. Alternatively, the composition may comprise a hostcell of the present invention as a source of the polypeptide havingendoglucanase activity in a fermentation process with the biomass. Inparticular, the polypeptides and host cells of the present invention maybe used to increase the value of processing residues (dried distillersgrain, spent grains from brewing, sugarcane bagasse, etc.) by partial orcomplete degradation of cellulose or hemicellulose. The host cell mayalso contain native or heterologous genes that encode other proteins andenzymes, mentioned above, useful in the processing of biomass.

In the methods of the present invention, any cellulosic material, suchas biomass, can be used. It is understood herein that the term“cellulosic material” encompasses lignocellulose. Biomass can include,but is not limited to, wood resources, municipal solid waste,wastepaper, crops, and crop residues (see, for example, Wiselogel etal., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork).

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

Three major classes of enzymes are used to breakdown cellulosic biomass:

(1) The “endo-1,4-beta-glucanases” or1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act randomlyon soluble and insoluble 1,4-beta-glucan substrates.

(2) The “exo-1,4-beta-D-glucanases” including both the 1,4-beta-D-glucanglucohydrolases (EC 3.2.1.74), which liberate D-glucose from1,4-beta-D-glucans and hydrolyze D-cellobiose slowly, andcellobiohydrolases (1,4-beta-D-glucan cellobiohydrolases, EC 3.2.1.91),which liberate D-cellobiose from 1,4-beta-glucans.

(3) The “beta-D-glucosidases” or beta-D-glucoside glucohydrolases (EC3.2.1.21), which act to release D-glucose units from cellobiose andsoluble cellodextrins, as well as an array of glycosides.

The polypeptides having endoglucanase activity of the present inventionare preferably used in conjunction with other cellulolytic proteins,e.g., exo-1,4-beta-D-glucanases and beta-D-glucosidases, to degrade thecellulose component of the biomass substrate, (see, for example, Brighamet al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.119-141, Taylor & Francis, Washington D.C.; Lee, 1997, Journal ofBiotechnology 56: 1-24).

The exo-1,4-beta-D-glucanases and beta-D-glucosidases may be produced byany known method known in the art (see, e.g., Bennett, J. W. and LaSure,L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991).

The optimum amounts of a polypeptide having endoglucanase activity andother cellulolytic proteins depends on several factors including, butnot limited to, the mixture of component cellulolytic proteins, thecellulosic substrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation). The term “cellulolytic proteins” isdefined herein as those proteins or mixtures of proteins shown as beingcapable of hydrolyzing or converting or degrading cellulose under theconditions tested.

In a preferred aspect, the amount of polypeptide having endoglucanaseactivity per g of cellulosic material is about 0.5 to about 50 mg,preferably about 0.5 to about 40 mg, more preferably about 0.5 to about25 mg, more preferably about 0.75 to about 20 mg, more preferably about0.75 to about 15 mg, even more preferably about 0.5 to about 10 mg, andmost preferably about 2.5 to about 10 mg per g of cellulosic material.

In another preferred aspect, the amount of cellulolytic proteins per gof cellulosic material is about 0.5 to about 50 mg, preferably about 0.5to about 40 mg, more preferably about 0.5 to about 25 mg, morepreferably about 0.75 to about 20 mg, more preferably about 0.75 toabout 15 mg, even more preferably about 0.5 to about 10 mg, and mostpreferably about 2.5 to about 10 mg per g of cellulosic material.

In the methods of the present invention, the composition may besupplemented by one or more additional enzyme activities to improve thedegradation of the cellulosic material. Preferred additional enzymes arehemicellulases, esterases (e.g., lipases, phospholipases, and/orcutinases), proteases, laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

The enzymes may be derived or obtained from any suitable origin,including, bacterial, fungal, yeast or mammalian origin. The term“obtained” means herein that the enzyme may have been isolated from anorganism which naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism, wherein the recombinantly producedenzyme is either native or foreign to the host organism or has amodified amino acid sequence, e.g., having one or more amino acids whichare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme which is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained recombinantly, such as by site-directed mutagenesis orshuffling.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be “substantially pure,” that is, free fromother components from the organism in which it is produced, that is, forexample, a host organism for recombinantly produced enzymes. In apreferred aspect, the enzymes are at least 75% (w/w), preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, even more preferably at least 98%, or mostpreferably at least 99% pure. In another preferred aspect, the enzyme is100% pure.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

The methods of the present invention may be used to process a cellulosicmaterial to many useful organic products, chemicals and fuels. Inaddition to ethanol, some commodity and specialty chemicals that can beproduced from cellulose include xylose, acetone, acetate, glycine,lysine, organic acids (e.g., lactic acid), 1,3-propanediol, butanediol,glycerol, ethylene glycol, furfural, polyhydroxyalkanoates, cis,cis-muconic acid, and animal feed (Lynd, L. R., Wyman, C. E., andGerngross, T. U., 1999, Biocommodity Engineering, Biotechnol. Prog., 15:777-793; Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212; and Ryu, D. D. Y., andMandels, M., 1980, Cellulases: biosynthesis and applications, Enz.Microb. Technol., 2: 91-102). Potential coproduction benefits extendbeyond the synthesis of multiple organic products from fermentablecarbohydrate. Lignin-rich residues remaining after biological processingcan be converted to lignin-derived chemicals, or used for powerproduction.

Conventional methods used to process the cellulosic material inaccordance with the methods of the present invention are well understoodto those skilled in the art. The methods of the present invention may beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Such an apparatus may include a batch-stirred reactor, a continuous flowstirred reactor with ultrafiltration, a continuous plug-flow columnreactor (Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of theenzymatic hydrolysis of cellulose: 1. A mathematical model for a batchreactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor(Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose byusing an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or areactor with intensive stirring induced by an electromagnetic field(Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y.,Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis usinga novel type of bioreactor with intensive stirring induced byelectromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol is combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews 66: 506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,malonic acid, oxalic acid, propionic acid, succinic acid, and xylonicacid); ketones (e.g., acetone); amino acids (e.g., aspartic acid,glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

The present invention further relates to methods of producing asubstance, comprising: (a) saccharifying a cellulosic material with acomposition comprising an effective amount of a polypeptide havingendoglucanase activity; (b) fermenting the saccharified cellulosicmaterial of step (a) with one or more fermentating microorganisms; and(c) recovering the substance from the fermentation. The compositioncomprising the polypeptide having endoglucanase activity may be in theform of a crude fermentation broth with or without the cells removed orin the form of a semi-purified or purified enzyme preparation or thecomposition may comprise a host cell of the present invention as asource of the polypeptide having endoglucanase activity in afermentation process with the biomass.

The substance can be any substance derived from the fermentation. In apreferred embodiment, the substance is an alcohol. It will be understoodthat the term “alcohol” encompasses a substance that contains one ormore hydroxyl moieties. In a more preferred embodiment, the alcohol isarabinitol. In another more preferred embodiment, the alcohol isbutanol. In another more preferred embodiment, the alcohol is ethanol.In another more preferred embodiment, the alcohol is glycerol. Inanother more preferred embodiment, the alcohol is methanol. In anothermore preferred embodiment, the alcohol is 1,3-propanediol. In anothermore preferred embodiment, the alcohol is sorbitol. In another morepreferred embodiment, the alcohol is xylitol. See, for example, Gong, C.S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred embodiment, the substance is an organic acid. Inanother more preferred embodiment, the organic acid is acetic acid. Inanother more preferred embodiment, the organic acid is acetonic acid. Inanother more preferred embodiment, the organic acid is adipic acid. Inanother more preferred embodiment, the organic acid is ascorbic acid. Inanother more preferred embodiment, the organic acid is citric acid. Inanother more preferred embodiment, the organic acid is2,5-diketo-D-gluconic acid. In another more preferred embodiment, theorganic acid is formic acid. In another more preferred embodiment, theorganic acid is fumaric acid. In another more preferred embodiment, theorganic acid is glucaric acid. In another more preferred embodiment, theorganic acid is gluconic acid. In another more preferred embodiment, theorganic acid is glucuronic acid. In another more preferred embodiment,the organic acid is glutaric acid. In another preferred embodiment, theorganic acid is 3-hydroxypropionic acid. In another more preferredembodiment, the organic acid is itaconic acid. In another more preferredembodiment, the organic acid is lactic acid. In another more preferredembodiment, the organic acid is malic acid. In another more preferredembodiment, the organic acid is malonic acid. In another more preferredembodiment, the organic acid is oxalic acid. In another more preferredembodiment, the organic acid is propionic acid. In another morepreferred embodiment, the organic acid is succinic acid. In another morepreferred embodiment, the organic acid is xylonic acid. See, forexample, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another preferred embodiment, the substance is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. In another more preferred embodiment, theketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

In another preferred embodiment, the substance is an amino acid. Inanother more preferred embodiment, the organic acid is aspartic acid. Inanother more preferred embodiment, the amino acid is glutamic acid. Inanother more preferred embodiment, the amino acid is glycine. In anothermore preferred embodiment, the amino acid is lysine. In another morepreferred embodiment, the amino acid is serine. In another morepreferred embodiment, the amino acid is threonine. See, for example,Richard, A., and Margaritis, A., 2004, Empirical modeling of batchfermentation kinetics for poly(glutamic acid) production and othermicrobial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred embodiment, the substance is a gas. In another morepreferred embodiment, the gas is methane. In another more preferredembodiment, the gas is H₂. In another more preferred embodiment, the gasis CO₂. In another more preferred embodiment, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Production of a substance from cellulosic material typically requiresfour major steps. These four steps are pretreatment, enzymatichydrolysis, fermentation, and recovery. Exemplified below is a processfor producing ethanol, but it will be understood that similar processescan be used to produce other substances, for example, the substancesdescribed above.

Pretreatment. In the pretreatment or pre-hydrolysis step, the cellulosicmaterial is heated to break down the lignin and carbohydrate structure,solubilize most of the hemicellulose, and make the cellulose fractionaccessible to cellulolytic enzymes. The heating is performed eitherdirectly with steam or in slurry where a catalyst may also be added tothe material to speed up the reactions. Catalysts include strong acids,such as sulfuric acid and SO₂, or alkali, such as sodium hydroxide. Thepurpose of the pre-treatment stage is to facilitate the penetration ofthe enzymes and microorganisms. Cellulosic biomass may also be subjectto a hydrothermal steam explosion pre-treatment (See U.S. PatentApplication No. 20020164730).

Saccharification. In the enzymatic hydrolysis step, also known assaccharification, enzymes as described herein are added to thepretreated material to convert the cellulose fraction to glucose and/orother sugars. The saccharification is generally performed instirred-tank reactors or fermentors under controlled pH, temperature,and mixing conditions. A saccharification step may last up to 200 hours.Saccharification may be carried out at temperatures from about 30° C. toabout 65° C., in particular around 50° C., and at a pH in the rangebetween about 4 and about 5, especially around pH 4.5. To produceglucose that can be metabolized by yeast, the hydrolysis is typicallyperformed in the presence of a beta-glucosidase.

Fermentation. In the fermentation step, sugars, released from thecellulosic material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to ethanol by a fermenting organism,such as yeast. The fermentation can also be carried out simultaneouslywith the enzymatic hydrolysis in the same vessel, again under controlledpH, temperature, and mixing conditions. When saccharification andfermentation are performed simultaneously in the same vessel, theprocess is generally termed simultaneous saccharification andfermentation or SSF.

Any suitable cellulosic substrate or raw material may be used in afermentation process of the present invention. The substrate isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention include cellulose-containingmaterials, such as wood or plant residues or low molecular sugars DP1-3obtained from processed cellulosic material that can be metabolized bythe fermenting microorganism, and which may be supplied by directaddition to the fermentation medium.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsaccording to the invention are able to ferment, i.e., convert, sugars,such as glucose, xylose, arabinose, mannose, galactose, oroligosaccharides directly or indirectly into the desired fermentationproduct. Examples of fermenting microorganisms include fungal organisms,such as yeast. Preferred yeast includes strains of the Saccharomycesspp., and in particular, Saccharomyces cerevisiae. Commerciallyavailable yeast include, e.g., Red Star®/™/Lesaffre Ethanol Red(available from Red Star/Lesaffre, USA) FALI (available fromFleischmann's Yeast, a division of Burns Philp Food Inc., USA),SUPERSTART (available from Alltech), GERT STRAND (available from GertStrand AB, Sweden) and FERMIOL (available from DSM Specialties).

In a preferred embodiment, the yeast is a Saccharomyces spp. In a morepreferred embodiment, the yeast is Saccharomyces cerevisiae. In anothermore preferred embodiment, the yeast is Saccharomyces distaticus. Inanother more preferred embodiment, the yeast is Saccharomyces uvarum. Inanother preferred embodiment, the yeast is a Kluyveromyces. In anothermore preferred embodiment, the yeast is Kluyveromyces marxianus. Inanother more preferred embodiment, the yeast is Kluyveromyces fragilis.In another preferred embodiment, the yeast is a Candida. In another morepreferred embodiment, the yeast is Candida pseudotropicalis. In anothermore preferred embodiment, the yeast is Candida brassicae. In anotherpreferred embodiment, the yeast is a Clavispora. In another morepreferred embodiment, the yeast is Clavispora lusitaniae. In anothermore preferred embodiment, the yeast is Clavispora opuntiae. In anotherpreferred embodiment, the yeast is a Pachysolen. In another morepreferred embodiment, the yeast is Pachysolen tannophilus. In anotherpreferred embodiment, the yeast is a Bretannomyces. In another morepreferred embodiment, the yeast is Bretannomyces clausenii (Philippidis,G. P., 1996, Cellulose bioconversion technology, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z.,Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho, N. W. Y., Chen, Z, Brainard, A.P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol. 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or another microorganism typically is added to the degradedcellulose or hydrolysate and the fermentation is ongoing for about 24 toabout 96 hours, such as about 35 to about 60 hours. The temperature istypically between about 26° C. to about 40° C., in particular at about32° C., and at about pH 3 to about pH 6, in particular around pH 4-5.

In a preferred embodiment, yeast or another microorganism is applied tothe degraded cellulose or hydrolysate and the fermentation is ongoingfor about 24 to about 96 hours, such as typically 35-60 hours. In apreferred embodiments, the temperature is generally between about 26 toabout 40° C., in particular about 32° C., and the pH is generally fromabout pH 3 to about pH 6, preferably around pH 4-5. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 5×10⁷ viable count per ml of fermentation broth. During anethanol producing phase the yeast cell count should preferably be in therange from approximately 10⁷ to 10¹⁰, especially around approximately2×10⁸. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process, Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients comprisingP, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery. The alcohol is separated from the fermented cellulosicmaterial and purified by conventional methods of distillation. Ethanolwith a purity of up to about 96 vol. % ethanol can be obtained, whichcan be used as, for example, fuel ethanol, drinking ethanol, i.e.,potable neutral spirits, or industrial ethanol.

For other substances, any method known in the art can be used including,but not limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing), differentialsolubility (e.g., ammonium sulfate precipitation), SDS-PAGE,distillation, or extraction.

In the methods of the present invention, the polypeptide havingendoglucanase activity and other cellulolytic protein(s) may besupplemented by one or more additional enzyme activities to improve thedegradation of the cellulosic material. Preferred additional enzymes arehemicellulases, esterases (e.g., lipases, phospholipases, and/orcutinases), proteases, laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

Propeptide and Signal Peptides

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein, wherein the gene is operably linked to one orboth of a first nucleotide sequence encoding a signal peptide comprisingor consisting of amino acids 1 to 16 of SEQ ID NO: 4, amino acids 1 to15 of SEQ ID NO: 6, amino acids 1 to 21 of SEQ ID NO: 8, or amino acids1 to 16 of SEQ ID NO: 10 and a second nucleotide sequence encoding apropeptide comprising or consisting of amino acids 17 to 24 of SEQ IDNO: 10, wherein the gene is foreign to the first and second nucleotidesequences

In a preferred aspect, the first nucleotide sequence comprises orconsists of nucleotides 19 to 69 of SEQ ID NO: 3. In another preferredaspect, the first nucleotide sequence comprises or consists ofnucleotides 39 to 83 of SEQ ID NO: 5. In another preferred aspect, thefirst nucleotide sequence comprises or consists of nucleotides 14 to 76of SEQ ID NO: 7. In another preferred aspect, the first nucleotidesequence comprises or consists of nucleotides 1 to 48 of SEQ ID NO: 9.In another preferred aspect, the second nucleotide sequence comprises orconsists of nucleotides 49 to 72 of SEQ ID NO: 9.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising (a) cultivating such a recombinant host cell under conditionssuitable for production of the protein; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Myceliophthora thermophila CBS 117.65, basidiomycete CBS 494.95,basidiomycete CBS 495.95, and Penicillium brasilianum strain IBT 20888(IBT Culture Collection of Fungi, Technical University of Denmark,Copenhagen, Denmark) were used as sources of the endoglucanase genes.Saccharomyces cerevisiae strain W3124 (MATa; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prc1::HIS3; prb1:: LEU2; cir⁺) was used forscreening of Myceliophthora thermophila CBS 117.65 expression librariesfor endoglucanase activity. Aspergillus oryzae HowB104(alpha-amylase-negative) was used for expression of the cel5a genes.

Media and Solutions

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of sodium chloride.

LB ampicillin medium was composed per liter of 10 g of tryptone, 5 g ofyeast extract, 5 g of sodium chloride, and 50 μg of ampicillin per ml(filter sterilized, added after autoclaving).

LB ampicillin plates were composed per liter of LB ampicillin medium and15 g of bacto agar.

YPD medium was composed of 1% yeast extract, 2% peptone, andfilter-sterilized 2% glucose added after autoclaving.

YPM medium was composed of 1% yeast extract, 2% peptone, andfilter-sterilized 2% maltodextrin added after autoclaving.

SC-URA medium with galactose was composed per liter of 100 ml of 10×Basal salts, 28 ml of 20% casamino acids without vitamins, 10 ml of 1%tryptophan, 3.6 ml of 5% threonine (filter sterilized, added afterautoclaving), and 100 ml of 20% galactose (filter sterilized, addedafter autoclaving).

SC-URA medium with glucose was composed per liter of 100 ml of 10× Basalsalts solution, 28 ml of 20% casamino acids without vitamins, 10 ml of1% tryptophan, 3.6 ml of 5% threonine (filter sterilized, added afterautoclaving), and 100 ml of 20% glucose (filter sterilized, added afterautoclaving).

10× Basal salts solution was composed per liter of 75 g of yeastnitrogen base, 113 g of succinic acid, and 68 g of NaOH.

SC-agar was composed per liter of SC-URA medium (with glucose orgalactose as indicated) and 20 g of agar.

0.1% AZCL HE cellulose SC agar plates with galactose were composed perliter of SC-URA medium with galactose, 20 g of agar, and 0.1% AZCL HEcellulose (Megazyme, Wicklow, Ireland).

BA medium was composed per liter of 10 g of corn steep liquor drymatter, 10 g of NH₄NO₃, 10 g of KH₂PO₄, 0.75 g of MgSO₄-7H₂O, 0.1 ml ofpluronic, and 0.5 g of CaCO₃. The pH was adjusted to 6.5 beforeautoclaving.

COVE plates were composed per liter of 342.3 g of sucrose, 25 g of Nobleagar, 20 ml of COVE salts solution, 10 mM acetamide, and 20 mM CsCl. Thesolution was adjusted to pH 7.0 before autoclaving.

COVE salts solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals.

COVE trace metals solution was composed per liter of 0.04 g ofNaB₄O7.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

TE was composed of 10 mM Tris pH 7.4 and 0.1 mM EDTA.

Example 1 Construction of Myceliophthora thermophila CBS 117.65 cDNAExpression Libraries in Saccharomyces cerevisiae

Myceliophthora thermophila CBS 117.65 was cultivated in 200 ml of BAmedium at 30° C. for five days at 200 rpm. Mycelia from the shake flaskculture were harvested by filtering the contents through a funnel linedwith Miracloth™ (CalBiochem, San Diego, Calif., USA). The mycelia werethen sandwiched between two Miracloth™ pieces and blotted dry withabsorbent paper towels. The mycelial mass was then transferred to Falcon1059 plastic centrifuge tubes and frozen in liquid nitrogen. Frozenmycelia were stored in a −80° C. freezer until use.

The extraction of total RNA was performed with guanidinium thiocyanatefollowed by ultracentrifugation through a 5.7 M CsCl cushion, andisolation of poly(A)+ RNA was carried out by oligo(dT)-celluloseaffinity chromatography, using the procedures described in WO 94/14953.

Double-stranded cDNA was synthesized from 5 μg of poly(A)+ RNA by theRNase H method (Gubler and Hoffman, 1983, Gene 25: 263-269, Sambrook etal., 1989, Molecular cloning: A laboratory manual, Cold Spring Harborlab., Cold Spring Harbor, N.Y., USA). The poly(A)⁺ RNA (5 μg in 5 μl ofDEPC (0.1% diethylpyrocarbonate)-treated water) was heated at 70° C. for8 minutes in a pre-siliconized, RNase-free Eppendorf tube, quenched onice, and combined in a final volume of 50 μl with reverse transcriptasebuffer composed of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol (DTT) (Bethesda Research Laboratories, Bethesda, Md.,USA), 1 mM of dATP, dGTP and dTTP, and 0.5 mM 5-methyl-dCTP (Pharmacia,Uppsala, Sweden), 40 units of human placental ribonuclease inhibitor(RNasin, Promega, Madison, Wis., USA), 1.45 μg of oligo(dT)₁₈-Not Iprimer (Pharmacia), and 1000 units of SuperScript II RNase H reversetranscriptase (Bethesda Research Laboratories). First-strand cDNA wassynthesized by incubating the reaction mixture at 45° C. for 1 hour.After synthesis, the mRNA:cDNA hybrid mixture was gel filtrated througha MicroSpin S-400 HR spin column (Pharmacia) according to themanufacturer's instructions.

After gel filtration, the hybrids were diluted in 250 μl of secondstrand buffer (20 mM Tris-HCl, pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂SO₄, 0.16 mM NAD) containing 200 μM of each dNTP, 60 units of E.coli DNA polymerase I (Pharmacia), 5.25 units of RNase H (Promega), and15 units of E. coli DNA ligase (Boehringer Mannheim, Manheim, Germany).Second strand cDNA synthesis was performed by incubating the reactiontube at 16° C. for 2 hours and an additional 15 minutes at 25° C. Thereaction was stopped by addition of EDTA to a final concentration of 20mM followed by phenol and chloroform extractions.

The double-stranded cDNA was precipitated at −20° C. for 12 hours byaddition of 2 volumes of 96% ethanol and 0.2 volume of 10 M ammoniumacetate, recovered by centrifugation at 13,000×g, washed in 70% ethanol,dried, and resuspended in 30 μl of Mung bean nuclease buffer (30 mMsodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35 mM DTT, 2%glycerol) containing 25 units of Mung bean nuclease (Pharmacia). Thesingle-stranded hair-pin DNA was clipped by incubating the reaction at30° C. for 30 minutes, followed by addition of 70 μl of 10 mM Tris-HCl-1mM EDTA pH 7.5, phenol extraction, and precipitation with 2 volumes of96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2 on ice for 30minutes.

The double-stranded cDNAs were recovered by centrifugation at 13,000×gand blunt-ended in 30 μl of T4 DNA polymerase buffer (20 mMTris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate,1 mM DTT) containing 0.5 mM of each dNTP and 5 units of T4 DNApolymerase (New England Biolabs, Ipswich, Mass., USA) by incubating thereaction mixture at 16° C. for 1 hour. The reaction was stopped byaddition of EDTA to a final concentration of 20 mM, followed by phenoland chloroform extractions, and precipitation for 12 hours at −20° C. byadding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH5.2.

After the fill-in reaction the cDNAs were recovered by centrifugation at13,000×g, washed in 70% ethanol, and dried. The cDNA pellet wasresuspended in 25 μl of ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mMMgCl₂, 10 mM DTT, 0.5 mM ATP) containing 2.5 μg of non-palindromic BstXI adaptors (Invitrogen, Carlsbad, Calif., USA), shown below, and 30units of T4 ligase (Promega), and then incubated at 16° C. for 12 hours.The reaction was stopped by heating at 65° C. for 20 minutes and thencooled on ice for 5 minutes.

5′-CTTTCCAGCACA-3′ (SEQ ID NO: 1) 3′-GAAAGGTC-5′ (SEQ ID NO: 2)

The adapted cDNA was digested with Not I, followed by incubation for 2.5hours at 37° C. The reaction was stopped by heating at 65° C. for 10minutes. The cDNAs were size-fractionated by gel electrophoresis on a0.8% SeaPlaque GTG low melting temperature agarose gel (CambrexCorporation, East Rutherford, N.J., USA) in 44 mM Tris Base, 44 mM boricacid, 0.5 mM EDTA (TBE) buffer to separate unligated adaptors and smallcDNAs. The cDNA was size-selected with a cut-off at 0.7 kb and rescuedfrom the gel by use of β-Agarase (New England Biolabs, Ipswich, Mass.,USA) according to the manufacturer's instructions and precipitated for12 hours at −20° C. by adding two volumes of 96% ethanol and 0.1 volumeof 3 M sodium acetate pH 5.2.

The directional, size-selected cDNA was recovered by centrifugation at13,000×g, washed in 70% ethanol, dried, and then resuspended in 30 μl of10 mM Tris-HCl-1 mM EDTA pH 7.5. The cDNAs were desalted by gelfiltration through a MicroSpin S-300 HR spin column according to themanufacturer's instructions. Three test ligations were carried out in 10μl of ligation buffer (30 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 10 mM DTT,0.5 mM ATP) containing 5 μl of double-stranded cDNA (reaction tubes #1and #2), 15 units of T4 ligase (Promega), and 30 ng (tube #1), 40 ng(tube #2), and 40 ng (tube #3, the vector background control) of BstXI-Not I cleaved pYES2.0 vector (Invitrogen, Carlsbad, Calif., USA). Theligation reactions were performed by incubation at 16° C. for 12 hours,then heating at 70° C. for 20 minutes, and finally adding 10 μl of waterto each tube. One μl of each ligation mixture was electroporated into 40μl of electrocompetent E. coli DH10B cells (Bethesda ResearchLaboratories) as described by Sambrook et al., 1989, supra.

The Myceliophthora thermophila CBS 117.65 cDNA library was establishedin E. coli DH10B consisting of pools. Each pool was made by spreadingtransformed E. coli on LB ampicillin plates, yielding 15,000-30,000colonies/plate after incubation at 37° C. for 24 hours. Twenty ml of LBampicillin medium was added to the plate and the cells were suspendedtherein. The cell suspension was shaken at 100 rpm in a 50 ml tube for 1hour at 37° C.

The resulting Myceliophthora thermophila CBS 117.65 cDNA libraryconsisted of approximately 10⁶ individual clones, with a vectorbackground of 1%. Plasmid DNA from some of the library pools wasisolated using a Plasmid Midi Kit (QIAGEN Inc., Valencia, Calif., USA),according to the manufacturer's instructions, and stored at −20° C.

Example 2 Screening of Myceliophthora thermophila CBS 117.65 ExpressionLibraries for Endoglucanase Activity

One ml aliquots of purified plasmid DNA (100 ng/ml) from some of thelibrary pools (Example 1) were transformed into Saccharomyces cerevisiaeW3124 by electroporation (Becker and Guarante, 1991, Methods Enzymol.194: 182-187) and the transformants were plated on SC agar containing 2%glucose and incubated at 30° C. In total, 50-100 plates containing250-400 yeast colonies were obtained from each pool.

After 3-5 days of incubation, the SC agar plates were replica platedonto a set of 0.1% AZCL HE cellulose SC URA agar plates with galactose.The plates were incubated for 2-4 days at 30° C. and endoglucanasepositive colonies were identified as colonies surrounded by a blue halo.

Example 3 Characterization of the Myceliophthora thermophila CBS 117.65cel5a Gene

Endoglucanase-expressing yeast colonies were inoculated into 20 ml ofYPD medium in 50 ml glass test tubes. The tubes were shaken at 200 rpmfor 2 days at 30° C. The cells were harvested by centrifugation for 10minutes at 3000 rpm in a Heraeus Megafuge 1.0R centrifuge with a75002252 rotor (Hanau, Germany).

DNA was isolated according to WO 94/14953 and dissolved in 50 μl ofdeionized water. The DNA was transformed into E. coli DH10B cells bystandard procedures according to Sambrook et al., 1989, supra. One E.coli transformant subsequently shown to contain the Myceliophthorathermophila CBS 117.65 cel5a gene was designated pCIC161 (FIG. 1) andused as the material for deposit of biological material. E. coli strainpCIC161 was deposited as E. coli NRRL B-30902 on Feb. 23, 2006.

Plasmid DNA was isolated from the E. coli transformants using standardprocedures according to Sambrook et al., 1989, supra. The full lengthcDNA sequence of the cel5a gene from Myceliophthora thermophila CBS117.65 was sequenced with a Taq DyeDeoxy Terminator Cycle Sequencing Kit(Perkin Elmer, Wellesley, Mass., USA) and synthetic oligonucleotideprimers using an Applied Biosystems ABI PRISM™ 377 DNA Sequencer (ABI,Foster City, Calif., USA) according to the manufacturer's instructions.

The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the Myceliophthora thermophila cel5a gene are shown inFIG. 2. The coding sequence is 1170 bp including the stop codon. Theencoded predicted protein contains 389 amino acids. The % G+C of thecoding region of the gene is 63.6% and the mature polypeptide codingregion is 63.6%. Using the SignalP program, version 3 (Nielsen et al.,1997, Protein Engineering 10:1-6), a signal peptide of 16 residues waspredicted. The predicted mature protein contains 373 amino acids with amolecular mass of 40.9 kDa.

Analysis of the deduced amino acid sequence of the cel5a gene with theInterproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17:847-848) showed that the CEL5A protein contained the core sequencetypical of a Family 5 glycosyl hydrolase, extending from approximatelyamino acid residue 77 to residue 350 of the predicted maturepolypeptide. The CEL5A protein also contained the sequence signature ofa type I fungal cellulose binding domain (CBMI). This sequence signatureknown as Prosite pattern PS00562 (Sigrist et al., 2002, Brief Bioinform.3: 265-274) was present from amino acid residue 8 to residue 35 of thepredicted mature polypeptide.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program ofEMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Myceliophthora thermophila gene encoding the CEL5Amature polypeptide shared 75% and 72% identity (excluding gaps) to thededuced amino acid sequences of two Family 5 glycosyl hydrolase proteinsfrom Neurospora crassa and Humicola insolens, respectively (accessionnumbers Q7SDR1 and Q12624, respectively).

Example 4 Expression of Myceliophthora thermophila CBS 117.65 Cel5a Genein Aspergillus oryzae

The Myceliophthora thermophila CBS 117.65 cel5a gene was excised fromthe pYES2.0 vector using Hind III and Xba I, and ligated into theAspergillus expression vector pHD414 (EP 238 023, WO 93/11249) usingstandard methods (Sambrook et al., 1989, supra). The Aspergillusexpression vector pHD414 is a derivative of p775 (EP 238 023). Theresulting plasmid was designated pA2C161 (FIG. 3).

Protoplasts of Aspergillus oryzae HowB104 were prepared as described inWO 95/02043. One hundred microliters of protoplast suspension was mixedwith 5-25 μg of the Aspergillus expression vector pA2C161 in 10 μl ofSTC composed of 1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCl₂) andfurther mixed with 5-25 μg of p3SR2, an Aspergillus nidulans amdS genecarrying plasmid (Christensen et al., 1988, Bio/Technology 6:1419-1422). The mixture was left at room temperature for 25 minutes. Twohundred microliters of 60% PEG 4000 (BDH, Poole, England) (polyethyleneglycol, molecular weight 4,000), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5was added and gently mixed and finally 0.85 ml of the same solution wasadded and gently mixed. The mixture was left at room temperature for 25minutes, centrifuged at 2,500×g for 15 minutes, and the pellet wasresuspended in 2 ml of 1.2 M sorbitol. This sedimentation process wasrepeated, and the protoplasts were spread on COVE plates. Afterincubation for 4-7 days at 37° C. spores were picked and spread in orderto isolate single colonies. This procedure was repeated and spores of asingle colony after the second reisolation were stored.

Each of the transformants was inoculated in 10 ml of YPM medium. After2-5 days of incubation at 30° C., 200 rpm, the supernatant was removed.Endoglucanase activity was identified by applying 20 μl of culture brothto 4 mm diameter holes punched out in a 0.1% AZCL HE cellulose SC-agarplate and incubation overnight at 30° C. Endoglucanase activity was thenidentified by a blue halo around a colony. Several transformant brothshad endoglucanase activity significantly greater than broth from anuntransformed Aspergillus oryzae background control, which demonstratedefficient expression of the CEL5A endoglucanase from Myceliophthorathermophila CBS 117.65 in Aspergillus oryzae.

Example 5 Construction of a Basidiomycete CBS 495.95 cDNA ExpressionLibrary in Saccharomyces cerevisiae

A cDNA library from basidiomycete CBS 495.95, consisting ofapproximately 10⁶ individual clones was constructed in E. coli asdescribed in Example 1, with a vector background of 1%.

Example 6 Screening of Basidiomycete CBS 495.95 cDNA ExpressionLibraries for Endoglucanase Activity

The screening of the cDNA library (Example 5) was performed as describedin Example 2.

Example 7 Characterization of a Cel5a Encoding Gene from BasidiomyceteCBS 495.95

Cloning of the cel5a gene from basidiomycete CBS 495.95 was carried outas described in Example 3. One E. coli transformant subsequently shownto contain a cel5a gene was designated pCIC453 (FIG. 4) and used as thematerial for deposit of biological material. E. coli strain pCIC453 wasdeposited as E. coli NRRL B-30903 on Feb. 23, 2006.

The basidiomycete CBS 495.95 cel5a gene was excised from pCIC453 usingHind III and Xba I and ligated into the Aspergillus expression vectorpHD414. The resulting plasmid was designated pA2C453 (FIG. 5).

The nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence(SEQ ID NO: 6) of the CBS 495.95 cel5a gene are shown in FIG. 6. Thecoding sequence is 1194 bp including the stop codon. The % G+C of thecoding region of the gene is 59.8% and the mature polypeptide codingregion is 60.1%. The encoded predicted protein contains 397 amino acids.Using the SignalP program, version 3 (Nielsen et al., 1997, supra), asignal peptide of 15 residues was predicted. The predicted matureprotein contains 382 amino acids with a molecular mass of 40.1 kDa.

Analysis of the deduced amino acid sequence of the cel5a gene with theInterproscan program (Zdobnov and Apweiler, 2001, supra) showed that theCEL5A protein contained the core sequence typical of a Family 5 glycosylhydrolase, extending from approximately residues 81 to 359 of thepredicted mature polypeptide. The CEL5A protein also contained thesequence signature of a type I fungal cellulose binding domain (CBMI).This sequence signature known as Prosite pattern PS00562 (Sigrist etal., 2002, supra) was present from amino acid residue 8 to residue 36 ofthe predicted mature polypeptide.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theCBS 495.95 gene encoding the CEL5A mature polypeptide shared 82% and 79%identity (excluding gaps) to the deduced amino acid sequences of twoFamily 5 glycosyl hydrolase proteins from Irpex lacteus and Trameteshirsuta, respectively (accession numbers Q5W7K4 and Q75UV6,respectively).

Example 8 Expression of Cel5a Gene from Basidiomycete CBS 495.95 inAspergillus oryzae

Expression of the cel5a gene from basidiomycete CBS 495.95, and analysisof endoglucanase activity was performed as described in Example 4.

Example 9 Construction of a Basidiomycete CBS 494.95 cDNA ExpressionLibrary in Saccharomyces cerevisiae

A cDNA library from basidiomycete CBS 494.95, consisting ofapproximately 10⁶ individual clones was constructed in E. coli asdescribed in Example 1, with a vector background of 1%.

Example 10 Screening of Basidiomycete CBS 494.95 cDNA ExpressionLibraries for Endoglucanase Activity

The screening of the cDNA library (Example 9) was performed as describedin Example 2.

Example 11 Characterization of a Cel5B Encoding Gene from BasidiomyceteCBS 494.95

Cloning of the cel5b gene from basidiomycete CBS 494.95 was carried outas described in Example 3. One E. coli transformant subsequently shownto contain the CBS 494.95 cel5b gene was designated pCIC486 (FIG. 7) andused as the material for deposit of biological material. E. coli strainpCIC486 was deposited as E. coli NRRL B-30904 on Feb. 23, 2006.

The basidiomycete CBS 494.95 cel5b gene was excised from the pYES2.0vector using Kpn I and Xho I and ligated into the Aspergillus expressionvector pHD423 (Lassen et al., 2001, Appl Environ Microbio) 67:4701-4707), a pHD414 derivative with a Kpn I site in the polylinker. Theresulting plasmid was designated pA2C486 (FIG. 8). The nucleotidesequence (SEQ ID NO: 7) and deduced amino acid sequence (SEQ ID NO: 8)of the CBS 494.95 cel5b gene are shown in FIG. 9. The coding sequence is1290 bp including the stop codon. The % G+C of the coding region of thegene is 56.0% G+C and the mature polypeptide coding region is 56.1%. Theencoded predicted protein contains 429 amino acids. Using the SignalPprogram, version 3 (Nielsen et al., 1997, supra), a signal peptide of 21residues was predicted. The predicted mature protein contains 408 aminoacids with a molecular mass of 43.1 kDa.

Analysis of the deduced amino acid sequence of the cel5b gene with theInterproscan program (Zdobnov and Apweiler, 2001, supra) showed that theCEL5B protein contained the core sequence typical of a Family 5 glycosylhydrolase, extending from approximately amino acid residue 106 toresidue 385 of the predicted mature polypeptide. The CEL5A protein alsocontained the sequence signature of a type I fungal cellulose bindingdomain (CBMI). This sequence signature known as Prosite pattern PS00562(Sigrist et al., 2002, supra) was present from amino acid residue 7 toresidue 34 of the predicted mature polypeptide.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman-Wunsch algorithm (Needleman and Wunsch,1970, supra) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theCBS 495.95 gene encoding the CEL5A mature polypeptide shared 69% and 67%identity (excluding gaps) to the deduced amino acid sequences of twoFamily 5 glycosyl hydrolase proteins from Irpex lacteus and Trameteshirsuta, respectively (accession numbers Q5W7K4 and Q75UV6,respectively).

Example 12 Expression of Cel5B from Basidiomycete CBS 494.95 inAspergillus oryzae

Expression of the cel5B gene from basidiomycete CBS 494.95, and analysisof endoglucanase activity was performed as described in Example 4.

Example 13 Purification of Recombinant Endoglucanases fromMyceliophthora thermophila CBS 117.65, basidiomycete CBS 494.95, andbasidiomycete CBS 495.95

The endoglucanases from Myceliophthora thermophila CBS 117.65,basidiomycete CBS 494.95, and basidiomycete CBS 495.95, producedrecombinantly in Aspergillus oryzae as described in Examples 4, 8, and12, were purified to homogeneity using a protocol essentially asdescribed by Otzen et al., 1999, Protein Sci. 8: 1878-87.

Protein concentration in the enzyme preparations was determined usingthe Bicinchoninic acid (BCA) Microplate Assay according to themanufacturer's instructions for a BCA Protein Assay Reagent Kit (PierceChemical Co., Rockford, Ill., USA).

Enzyme dilutions were prepared fresh before each experiment from stockenzyme solutions, which were stored at −20° C.

Example 14 Isolation of Genomic DNA from Penicillium brasilianum IBT20888

Spores of Penicillium brasilianum strain IBT 20888 were propagated onrice according to Carlsen, 1994, Ph.D. thesis, Department ofBiotechnology, The Technical University of Denmark. The spores wererecovered with 20 ml of 0.1% Tween 20 and inoculated at a concentrationof 1×10⁶ spores per ml into 100 ml of Mandels and Weber medium (Mandelsand Weber, 1969, Adv. Chem. Ser. 95: 394-414) containing 1% glucosesupplemented per liter with 0.25 g of yeast extract and 0.75 g ofBactopeptone in a 500 ml baffled shake flask. The fungal mycelia wereharvested after 24 hours of aerobic growth at 30° C., 150 rpm.

Mycelia were collected by filtration through a Nalgene DS0281-5000filter (Nalge Nunc International Corporation, Rochester, N.Y., USA)until dryness and frozen in liquid nitrogen. The frozen mycelia wereground to a powder in a dry ice chilled mortar and distributed to ascrew-cap tube. The powder was suspended in a total volume of 40 ml of50 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid)-NaOH pH 11buffer containing 0.5% lithium dodecyl sulfate and 0.5 mM EDTA. Thesuspension was placed at 60° C. for 2 hours and periodically resuspendedby inversion. To the suspension was added an equal volume ofphenol:chloroform (1:1 v/v) neutralized with 0.1 M Tris base, and thetube was mixed on a rotating wheel at 37° C. for 2 hours. Aftercentrifugation at 2500 rpm for 10 minutes in a Sorvall H1000B rotor, theaqueous phase (top phase) was re-extracted again with phenol:chloroform(1:1 v/v) and centrifuged at 15,000×g for 5 minutes. The aqueous phasefrom the second extraction was brought to 2.5 M ammonium acetate (stock10 M) and placed at −20° C. until frozen. After thawing, the extract wascentrifuged at 15,000×g for 20 minutes in a cold rotor. The pellet(primarily rRNA) was discarded and the nucleic acids in the supernatantwere precipitated by addition of 0.7 volumes of isopropanol. Aftercentrifugation at 15,000×g for 15 minutes, the pellet was rinsed threetimes with 5 ml of 70% ethanol (without resuspension), air-dried almostcompletely, and dissolved in 1.0 ml of 0.1× TE. The dissolved pellet wastransferred to two 1.5 ml microfuges tubes. The nucleic acids wereprecipitated by addition of ammonium acetate (0.125 ml) to 2.0 M andethanol to 63% (1.07 ml) and centrifuged at maximum speed for 10 minutesin a Sorvall MC 12V microcentrifuge (Kendro Laboratory Products,Asheville, N.C., USA). The pellet was rinsed twice with 70% ethanol,air-dried completely, and dissolved in 500 μl of 0.1× TE.

Example 15 Preparation of a Genomic DNA Library of Penicilliumbrasilianum IBT 20888

Genomic libraries were constructed using a TOPO Shotgun Subcloning Kit(Invitrogen, Carlsbad, Calif., USA). Briefly, total cellular DNA wassheared by nebulization under 10 psi nitrogen for 15 seconds andsize-fractionated on 1% agarose gels using 40 mM Tris base-20 mM sodiumacetate-1 mM disodium EDTA (TAE) buffer. DNA fragments migrating in thesize range 3-6 kb were excised and eluted using a MiniElute™ GelExtraction Kit (QIAGEN Inc, Valencia, Calif., USA). The eluted fragmentswere size-fractionated again using a 1% agarose gel as above and DNAfragments migrating in the size range 3-6 kb were excised and elutedusing a MiniElute™ Gel Extraction Kit.

The eluted DNA fragments were blunt end repaired and dephosphorylatedusing shrimp alkaline phosphatase (Roche Applied Science, Manheim,Germany). The blunt end DNA fragments were cloned into thepCR4Blunt-TOPO vector (Invitrogen, Carlsbad, Calif., USA) according tothe manufacturer's instructions, transformed into electrocompetent E.coli TOP10 cells by electroporation, and plated on LB ampicillin plates.The electroporation resulted in 15,300 clones.

Example 16 Purification of Native Cel5C Endoglucanase from Penicilliumbrasilianum IBT 20888

The endoglucanase was purified and assayed as described in Jorgensen etal., 2003 (Enzyme Microb. Technol. 32: 851-861). The substrate wasazo-carboxymethyl cellulose (Megazyme International Ireland Ltd., Bray,Ireland) and the incubation time was 15 minutes. Purified enzyme wasstored frozen at −20° C.

Example 17 N-terminal Sequencing of CEL5C Endoglucanase from Penicilliumbrasilianum IBT 20888

The purified sample of Penicillium brasilianum CEL5C endoglucanase(Example 16) was thawed. A 100 μl aliquot of the sample was added to 100μl of SDS-PAGE sample buffer (4 ml of 0.5 M TRIS-HCl, pH 6.8, 20 ml of10% SDS, 20 ml of glycerol (87%), 56 ml of Milli-Q® ultrapure water, and15 grains of bromophenol blue) in an Eppendorf tube and heated to 95° C.for 4 minutes. Following heating four 20 μl aliquots of the dilutedsample were applied separately to a precast 4-20% SDS polyacrylamide gel(Invitrogen, Carlsbad, Calif., USA). In addition to the four lanescontaining the sample, a Mark 12 protein standard mixture (Invitrogen,Carlsbad, Calif., USA) was applied to the gel.

The gel was run in an Xcell SureLock™ gel apparatus (Invitrogen,Carlsbad, Calif., USA) for 90 minutes with initial power settings of 40mA at maximum 135 V. Following electrophoresis the gel was incubated for5 minutes in a blotting solution consisting of 10 mM CAPS pH 11containing 6% methanol. A ProBlott membrane (Applied Biosystems, FosterCity, Calif., USA) was wetted for 1 minute in pure methanol before beingplaced in the blotting solution for 5 minutes in order to saturate themembrane with 10 mM CAPS pH 11 containing 6% methanol.

Electroblotting was carried out in a Semi Dry Blotter II apparatus(KemEnTec, Copenhagen, DK) as follows. Six pieces of Whatman no. 1 paperwetted in the blotting solution were placed on the positive electrode ofthe blotting apparatus followed by the ProBlott membrane, thepolyacrylamide gel, and six pieces of Whatman no. 1 paper wetted inblotting solution. The blotting apparatus was assembled thereby puttingthe negative electrode in contact with the upper stack of Whatman no. 1paper. A weight of 11.3 kg was placed on top of the blotting apparatus.The electroblotting was performed at a current of 175 mA for 180minutes.

Following the electroblotting the ProBlott membrane was stained for 1minute in 0.1% (w/v) Coomassie Brilliant Blue R-250 dissolved in 60%methanol, 1% acetic acid, 39% H₂O. Destaining of the ProBlott membranewas performed in 40% aqueous methanol for 5 minutes before the membranewas rinsed in deionized water. Finally the ProBlott membrane wasair-dried.

For N-terminal amino acid sequencing two pieces of the ProBlott membraneconsisting of a 65 kDa band were cut out and placed in the blottingcartridge of an Applied Biosystems Procise Protein Sequencer (AppliedBiosystems, Foster City, Calif., USA). The N-terminal sequencing wascarried out using the method run file for PVDF membrane samples (Pulsedliquid PVDF) according to the manufacturer's instructions.

The N-terminal amino acid sequence was deduced from the resultingchromatograms by comparing the retention time of the peaks in thechromatograms to the retention times of the PTH-amino-acids in thestandard chromatogram.

The N-terminal amino acid sequence of the purified Penicilliumbrasilianum CEL5C endoglucanase was determined directly using a Procise494 HT Sequencing System (Applied Biosystems, Foster City, Calif., USA).The N-terminal sequence was determined to beAla-Ser-Ser-Phe-Val-Trp-Phe-Gly-Thr-Ser-Glu-Ser-Gly-Ala-Glu-Phe-Gly-Asn-Gln-Asn(amino acids 25 to 44 in SEQ ID NO: 10).

Example 18 PCR Amplification of the cel5c Endoglucanase Gene fromPenicillium brasilianum IBT 20888

Based on the N-terminal amino acid sequence of the purified Penicilliumbrasilianum endoglucanase, a forward primer was designed using theCODEHOP strategy (Rose et al., 1998, Nucleic Acids Res. 26: 1628-35).From database information on other endoglucanases, two reverse primerswere designed as shown below using the CODEHOP strategy.

Forward primer, Fwd: (SEQ ID NO: 11) 5′-TTCGGTACCTCTGAGTCTGGNGCNGARTT-3′Reverse primer, Rev1: (SEQ ID NO: 12)5′-TGATCCATATCGTGGTACTCGTTRTTNGTRTCRAA-3′ Reverse primer, Rev2:(SEQ ID NO: 13) 5′-CCGTTGTAGCGACCGTARTTRTGNGGRTC-3′where R=A or G, Y═C or T, K=G or T and N=A, C, G or T

Amplification reactions (30 μl) were prepared using approximately 1 μgof Penicillium brasilianum genomic DNA as template. In addition, eachreaction contained the following components: 30 μmol of the forwardprimer, 30 μmol of the reverse primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1× AmpliTaq polymerase buffer (Applied Biosystems, FosterCity, Calif., USA), and 0.5 unit of AmpliTaq polymerase (5.0 U/μl,Applied Biosystems, Foster City, Calif., USA). The reactions wereincubated in a Robocycler (Stratagene, La Jolla, Calif., USA) programmedfor 1 cycle at 96° C. for 3 minutes and at 72° C. for 3 minutes; 34cycles each at 95° C. for 0.5 minute, 56° C. for 0.5 minutes, and 72° C.for 1.5 minutes; 1 cycle at 72° C. for 7 minutes; and a soak cycle at 6°C. Taq polymerase was added at 72° C. in the first cycle.

PCR reaction products were separated on a 2% agarose gel (Amresco,Solon, Ohio, USA) using TAE buffer. A band of approximately 650 bp (Fwdand Rev1 primers) and bands of approximately 320 and 380 bp (Fwd andRev2 primers) were excised from the gel and purified using a MiniElute™Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions. The purified PCR products were subsequentlyanalyzed by DNA sequencing. The 320 bp product was found to encode aportion of a glycosyl hydrolase Family 5 polypeptide that was designatedCEL5C.

Example 19 Screening of Genomic Library of Penicillium brasilianum IBT20888

Colony lifts of the library described in Example 15 were performed(Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y.) and the DNA wascross-linked onto Hybond N+ membranes (Amersham, Arlington Heights,Ill., USA) for 2 hours at 80° C. The membranes from the colony liftswere pre-wetted using 0.2×SSC (30 mM NaCl, 3 mM sodium citrate), 0.2%SDS. The pre-wetted filters were placed in a beaker with 7.5 ml of 6×SSPE (0.9 M NaCl, 0.06 M NaH₂PO₄, and 6 mM EDTA), 7% SDS) per filter at68° C. in a shaking water bath for 30 minutes.

Approximately 40 ng of the PCR product described in Example 18 wererandom-primer labeled using a Stratagene Prime-It II Kit (Stratagene, LaJolla, Calif.) according to the manufacturer's instructions. Theradiolabeled gene fragment was separated from unincorporated nucleotideusing a MinElute PCR Purification Kit (QIAGEN Inc., Valencia, Calif.,USA).

The radioactive probe was denatured by adding 5.0 M NaOH to a finalconcentration of 0.5 M, and added to the hybridization solution at anactivity of approximately 0.5×10⁶ cpm per ml of hybridization solution.The mixture was incubated for 10 hours at 68° C. in a shaking waterbath. Following incubation, the membranes were washed three times in0.2× SSC, 0.2% SDS at 68° C. The membranes were then dried on blottingpaper for 15 minutes, wrapped in SaranWrap™ and exposed to X-ray filmovernight at −80° C. with intensifying screens (Kodak, Rochester, N.Y.,USA).

Colonies producing hybridization signals with the probe were inoculatedinto 1 ml of LB ampicillin medium and cultivated overnight at 37° C.Dilutions of each solution were made and 100 μl were plated onto LBampicillin plates. The dilution for each positive that produced about 40colonies per plate was chosen for secondary lifts. The lifts wereprepared, hybridized, and probed as above. Two colonies from eachpositive plate were inoculated into 3 ml of LB ampicillin medium andcultivated overnight at 37° C.

Miniprep DNA was prepared from each colony using a Bio Robot 9600(QIAGEN Inc, Valencia, Calif., USA) according to the manufacturer'sprotocol. The size of each insert was determined by Eco RI restrictionand agarose gel electrophoresis. Two clones each contained anapproximately 5.5 kb insert. Sequencing revealed that the clones wereidentical, and they were hereafter referred to as pKKAH1 (see Example20).

Example 20 Characterization of the Cel5C Genomic Sequence Encoding theCEL5C Endoglucanase from Penicillium brasilianum IBT 20888

DNA sequencing of the Penicillium brasilianum endoglucanase gene frompKKAH1 was performed with an Applied Biosystems Model 3700 Automated DNASequencer (Applied Biosystems, Foster City, Calif., USA) using theprimer walking technique with dye-terminator chemistry (Giesecke et al.,1992, J. Virol. Methods 38: 47-60).

The nucleotide sequence (SEQ ID NO: 9) and deduced amino acid sequence(SEQ ID NO: 10) of the Penicillium brasilianum cel5c gene are shown inFIGS. 10A and 10B. The genomic coding sequence of 1471 bp (includingstop codon) encodes a polypeptide of 421 amino acids, interrupted by 4introns of 51 bp (89-139 bp), 47 bp (352-398 bp), 55 bp (464-518 bp),and 52 bp (617-668 bp). The % G+C content of the coding region of thegene is 51.2% and the mature polypeptide coding region is 50.8%. Usingthe SignalP software program (Nielsen et al., 1997, Protein Engineering10:1-6), a signal peptide of 16 residues was predicted. Based on theN-terminal sequence of the endoglucanase, residues 17 through 24 appearto constitute a pro-region that is proteolytically cleaved duringmaturation. The predicted mature protein contains 397 amino acids andhas a predicted mass of 42.6 kDa.

Analysis of the deduced amino acid sequence of the cel5c gene with theInterproscan program (Zdobnov and Apweiler, 2001, supra) showed that theCEL5C protein contained the core sequence typical of a Family 5 glycosylhydrolase, extending from approximately residues 32 to 307 of thepredicted full-length polypeptide. The CEL5C protein also contained thesequence signature of a type I fungal cellulose binding domain (CBMI).This sequence signature known as Prosite pattern PS00562 (Sigrist etal., 2002, supra) was present from amino acid residue 393 to residue 420of the predicted polypeptide.

A comparative pairwise global alignment of amino acid sequences inpublic databases was determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, supra) as implemented in the Needle programof EMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the Penicillium brasilianum cel5c gene encoding the CEL5Cmature polypeptide shared 74.9% and 74.5% identity (excluding gaps) tothe deduced amino acid sequences of two predicted Family 5 glycosylhydrolase proteins from Neosartorya fischeri and Aspergillus fumigatus,respectively (accession numbers A1DAP7 and Q4WM09, respectively).

Example 21 Construction of an Aspergillus oryzae Expression Plasmid forthe cel5c Endoglucanase Gene from Penicillium brasilianum IBT 20888

The Aspergillus expression plasmid pJaL721 (WO 03/008575) consists of anexpression cassette based on the Aspergillus niger neutral amylase IIpromoter fused to the Aspergillus nidulans triose phosphate isomerasenon-translated leader sequence (NA2-tpi) and the Aspergillus nigeramyloglycosidase terminator. Also present on the plasmid is theselective marker amdS from Aspergillus nidulans enabling growth onacetamide as sole nitrogen source and the URA3 marker from Saccharomycescerevisiae enabling growth of the pyrF defective Escherichia coli strainDB6507 (ATCC 35673). Transformation into E. coli DB6507 was performedusing the Saccharomyces cerevisiae URA3 gene as selective marker asdescribed below.

E. coli DB6507 was made competent by the method of Mandel and Higa,1970, J. Mol. Biol. 45: 154. Transformants were selected on solid M9medium (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.)supplemented per liter with 1 g of casamino acids, 500 μg of thiamine,and 10 mg of kanamycin.

The endoglucanase gene was cloned into pJaL721 as described below. Thecel5c endoglucanase gene from Penicillium brasilianum was amplified byPCR using the following two oligonucleotide primers:

Forward PCR: (SEQ ID NO: 14)5′-AATTGGATCCACCATGAAATACCCTCTACTCCTGGCAAC-3′ Reverse PCR:(SEQ ID NO: 15) 5′- TTAACTCGAGTTACAGACACTGCGAATAATACGCATTC-3′

To facilitate cloning a restriction enzyme site was inserted into the 5′end of each primer where the forward primer contained a Bam HI site andthe reverse primer contained an Xho I site.

Genomic DNA prepared as in Example 14 was used as template in the PCRreaction. The reaction was performed in a volume of 50 μl containing 1.0unit of Phusion (Finnzymes Oy, Espoo, Finland), 1× Phusion buffer HF(Finnzymes Oy, Espoo, Finland), 500 ng of genomic, 250 μM of each dNTP,and 50 μmol of each of the two primers described above. Theamplification was carried out in a PTC-220 DNA Engine Dyad PeltierThermal Cycler (MJ Research, Inc., Waltham, Mass., USA) programmed for 1cycle at 95° C. for 5 minutes; 24 cycles each at 94° C. for 0.5 minute,58° C. for 0.5 minute, and 68° C. for 4.0 minutes; and 1 cycle at 68° C.for 15 minutes. The hot start PCR technique (Chou et al., 1992, NucleicAcids Res. 20: 1717) was used and the Phusion polymerase was added after1 minute of the first cycle.

The PCR reaction produced a single DNA fragment of approximately 1500 bpin length. The fragment was digested with Bam HI and Xho I and isolatedby agarose gel electrophoresis, purified, and cloned into pJaL721digested with Bam HI and Xho I, resulting in a plasmid designated pKBK03(FIG. 11). The sequence of the endoglucanase gene in pKBK03 was verifiedby sequencing with an Applied Biosystems 3730xl DNA Analyzer.

In order to create a plasmid for deposit of biological material, pKBK03was digested with Bam HI and Xho I and purified. The fragment was bluntend repaired using Klenow enzyme (Roche Applied Science, Manheim,Germany) for 30 minutes at 25° C. Plasmid pUC13 was digested with Sma Iand dephosphorylated with Calf Intestinal Protease (Roche AppliedScience, Manheim, Germany; CIP) for 1 hour at 37° C. and the CIP wasinactivated by heating the sample to 80° C. for 15 minutes.

The blunt end repaired fragment and the dephosphorylated pUC13 fragmentwere ligated overnight at 16° C. using T4 DNA ligase (Roche AppliedScience, Manheim, Germany). A 0.25 μg sample of the ligated product wastransformed into Escherichia coli DH5α (Invitrogen, Carlsbad, Calif.,USA). After incubation overnight at 37° C. on LB ampicillin plates,transformants were transferred to 2 ml of LB medium and incubated at 37°C. A plasmid designated pPBCel5C (FIG. 12) was purified using JetquickPlasmid Miniprep (Genomed, Löhne, Germany). The sequence of theendoglucanase gene was verified by sequencing with an Applied Biosystems3730xl DNA Analyzer. E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.)containing plasmid pPBCel5C (strain designation PBCel5C) were depositedwith the Agricultural Research Service Patent Culture Collection,Northern Regional Research Center, 1815 University Street, Peoria, Ill.,61604, as NRRL B-30900N, with a deposit date of Feb. 23, 2006.

Example 22 Expression of the Penicillium brasilianum IBT 20888 CEL5Cendoglucanase in Aspergillus oryzae

Aspergillus oryzae BECh2 (WO 00/30322) was transformed with 5 μg ofpKBK03 as described by Christensen et al., 1988, Biotechnology 6:1419-1422.

Transformants were cultivated in 50 ml tubes for 4 days at 30° C. in 10ml of YPM medium. The whole broths were centrifuged at 12,100×g and thesupernatants removed. The supernatants were analyzed by SDS-PAGE using aCriterion XT Precast Gel, 10% Bis-Tris gel in a XT MES buffer (BioRadLaboratories, Hercules, Calif., USA) according to the manufacturer'sinstructions. A 10 μl volume of supernatant was mixed with 9 μl ofsample buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, and 4.6% SDS), and1 μl of 1 M dithiothreitol, and heated to 96° C. for 5 minutes. In 16out of 20 supernatants, one band of approximately 65 kDa was visible inthe range of the standards 35 kDa to 150 kDa by SDS-PAGE. Thesupernatants resulting in a visible band after SDS-PAGE also containedendoglucanase activity, assayed as described in Example 3. The higherthe intensity of the band, the higher endoglucanase activity measured inthe same supernatant.

One transformant was designated Aspergillus oryzae KBK03.

Example 23 Production and Purification of Recombinant PenicilliumBrasilianum

IBT 20888 CEL5C Endoglucanase

Aspergillus oryzae transformant KBK03 was grown in twenty 500 ml shakeflasks with 200 ml of YPM medium.

The biomass was removed from 4.0 liters of fermentation broth bycentrifugation and filtration. SDS-PAGE analysis was performed asdescribed in Example 9. The endoglucanase solution was loaded onto a XK50 column (Amersham Biosciences, Uppsala, Sweden) containing 110 g ofAvicel Ph 101 (Merck KGaA, Darmstadt, Germany) pre-equilibrated with 25mM Tris pH 7.5 prior to loading and the bound enzyme was eluted with 25mM Tris, 1% triethanolamine at pH 11.6. Elution of the endoglucanase wasmonitored at 280 nm. The eluted protein containing fractions were pooledimmediately and the pH adjusted to 7.5. Fractions containing theendoglucanase were pooled.

The protein content was determined from the absorbance at 280 nm and theextinction coefficient calculated from the primary structure of theendoglucanase.

The purification was followed by SDS-PAGE. The samples were boiled for 2minutes with an equal volume of 2× sample buffer and ⅕ volume of 1% PMSFand loaded onto a 4-20% Tris-glycine gel (Invitrogen, Carlsbad, Calif.,USA). The gel was stained with GelCode Blue Stain Reagent (Pierce,Rockford, Ill., USA) and destained with water. SDS-PAGE revealed oneband of approximately 65 kDa.

Example 24 Characterization of Purified Recombinant Penicilliumbrasilianum IBT 20888 CEL5C endoglucanase

The purified recombinant Penicillium brasilianum CEL5C endoglucanasedescribed in Example 23 was characterized with regard to pH optimum,temperature optimum, and temperature stability. The endoglucanaseactivity was measured as described in Example 16 at temperatures from20° C. to 80° C. and at pH values of 3.0 to 10.0. The purifiedendoglucanase was diluted in Milli-Q® ultrapure water (Millipore,Billerica, Mass., USA) to ensure that activity was within the standardcurve. For the pH optimum, the substrate was dissolved inBritton-Robinson buffer (50 mM boric acid, 50 mM acetic acid, 50 mMphosphoric acid) adjusted to the desired pH. The temperature stabilitywas determined for 20 hours at 50° C. in the pH range from 4.0 to 6.0.All experimental assays were performed in duplicate.

The results of the pH optimum determination is shown in FIG. 13. Theoptimum pH was close to 4.0 at 50° C. with very little activity at pH3.0 and approximately 80% of peak activity at pH 5.0.

The results of the temperature optimum determination is shown in FIG.14. The temperature optimum at pH 4.8 was approximately 70° C. with morethan 75% of peak activity from 60° C. to 80° C.

The results of the temperature stability determination is shown in FIG.15. When pre-incubated in the absence of substrate for 20 hours at 25°C. and 50° C. in the pH range from 4.0 to 6.0, the endoglucanaseretained more than 80% of its starting activity.

Example 25 Preparation of Substrates

Pretreated corn stover (PCS) was prepared by the U.S. Department ofEnergy

National Renewable Energy Laboratory (NREL) using dilute sulfuric acid.The following conditions were used for the pretreatment: 1.4 wt %sulfuric acid at 165° C. and 107 psi for 8 minutes. Compositionalanalysis was performed at NREL. Cellulose and hemicellulose weredetermined by a two-stage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography (HPLC)using NREL Standard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid (NREL Standard Analytical Procedure #003).Water-insoluble solids in the pretreated corn stover (PCS) weredetermined to be 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.

The PCS was washed with large volume of deionized water on a Kimaxfunnel with a glass filter of coarse porosity (Fisher Scientific,Pittsburg, Pa., USA). Water-washed PCS was milled in a coffee grinderand additionally washed with deionized water on a 22 μm Millipore Filterwith a 6P Express Membrane (Millipore, Bedford, Mass., USA). Dry weightof the milled PCS was 32.4%.

A 10 mg/ml stock suspension of phosphoric acid-swollen cellulose (PASC)in deionized water was prepared using the following procedure. Onehundred and fifty ml of ice-cold 85% o-phosphoric acid was added to 5 gof Avicel PH101 (FMC Corp., Philadelphia, Pa., USA) moistened withwater. The suspension was slowly stirred in an ice bath for one hour,and 100 ml of ice-cold acetone was added to the suspension at constantstirring. The slurry was transferred to a Kimax funnel with a glassfilter of coarse porosity, washed three times with 100 ml of ice-coldacetone, and drained as completely as possible after each wash. Finally,the slurry was washed twice with 500 ml of water, and again drained ascompletely as possible after each wash. The PASC was mixed with water toa total volume of 500 ml. Sodium azide was added to a finalconcentration of 0.02% to prevent microbial growth. The slurry washomogenized using a blender and stored at 4° C. for up to one month.

Carboxymethylcellulose (CMC, sodium salt, type 7L2) with an averagedegree of substitution (DS) of 0.7 was obtained from Aqualon Division ofHercules Inc., Wilmington, Del., USA. A 6.25 mg/ml solution of CMC in 50mM sodium acetate pH 5.0 was prepared by slowly adding CMC to thevigorously agitated buffer. The slurry was heated to approximately 60°C. under continuous stirring until the CMC was completely dissolved.

Bacterial cellulose (BC) was prepared from Nata de Coco, a food-gradecommercial cellulose (Fujicco Co., Kobe, Japan), as described in Boissetet al., 1999, Biochemical Journal, 340: 829-835. A 1 mg/ml suspension ofbacterial cellulose in deionized water with 0.01% (w/v) sodium azide wasstored at 4° C.

Avicel PH101 was obtained from FMC Corporation, Philadelphia, Pa., USA.

Xylan from birchwood was obtained from Sigma, St. Louis, Mo., USA.Xyloglucan from Tamarind seed (amyloid, lot 00401), wheat arabinoxylan(medium viscosity, 27 cSt, lot 90601), 1,4-beta-D-mannan (borohydridereduced, Man:Gal=97:3, degree of polymerization DP˜15, lot 90302), andcarob galactomannan (low viscosity, borohydride reduced, lot 90301) wereobtained from Megazyme, Bray, Ireland.

Example 26 p-Hydroxybenzoic Acid Hydrazide Assay for Determination ofReducing Sugars

Reducing sugars (RS) were determined by a p-hydroxybenzoic acidhydrazide

(PHBAH) assay (Lever, 1972, Anal. Biochem. 47: 273-279), which wasadapted to a 96-well microplate format.

A 90-μl aliquot of the diluted sample was placed into each well of a96-well conical-bottomed microplate (Costar, clear polycarbonate,Corning Inc., Acton, Mass., USA). The assay was initiated by adding 60μl of 1.25% PHBAH in 2% sodium hydroxide to each well. The uncoveredplate was heated on a custom-made heating block for 10 minutes at 95° C.Following heating, the microplate was cooled to room temperature, and 35μl of deionized water was added to each well. A 100 μl aliquot wasremoved from each well and transferred to a flat-bottomed 96-well plate(Costar, medium binding polystyrene, Corning Inc., Acton, Mass., USA).The absorbance at 410 nm (A₄₁₀) was measured using a SpectraMAXMicroplate Reader (Molecular Devices, Sunnyvale, Calif., USA). The A₄₁₀value was translated into glucose equivalents using a standard curve.

The standard curve was obtained with six glucose standards (0.005,0.010, 0.025, 0.050, 0.075, and 0.100 mg/ml), which were treatedsimilarly to the samples. Glucose standards were prepared by diluting 10mg/ml stock glucose solution with deionized water.

For all substrates except for xylan and arabinoxylan, the degree ofconversion (%) was calculated using the following equation:Conversion_((%))=RS_((mg/ml))×100×162/(Initial substrateconcentration_((mg/ml))×180)=RS_((mg/ml))×100/(Initial substrateconcentration_((mg/ml))×1.111)

For xylan and arabinoxylan, percent of substrate hydrolyzed to RS wascalculated using the following equation:Conversion_((%))=RS_((mg/ml))×100×132/(Initial substrateconcentration_((mg/ml))×150)=RS_((mg/ml))×100/(Initial substrateconcentration_((mg/ml))×1.136)

In these equations, RS is the concentration of reducing sugars insolution measured in glucose equivalents (mg/ml), and the factors 1.111and 1.136 reflect the weight gain in converting correspondingpolysaccharides to hexose (MW 180) or pentose (MW 150) sugars.

Example 27 Relative Activity of Endoglucanases onCarboxymethyl-Cellulose at 50° C.

Table 1 shows the relative activity of the purified endoglucanases fromMyceliophthora thermophila CBS 117.65, basidiomycete CBS 494.95, andbasidiomycete CBS 495.95 toward the soluble sodium salt ofcarboxymethylcellulose (CMC). The relative activity is shown aspercentage of the activity of basidiomycete CBS 495.95 endoglucanase.The activity was determined by measuring the concentration of reducingsugars (RS) produced from CMC (5 mg/ml) after 30 minutes of hydrolysisin 50 mM sodium acetate pH 5.0 at 50° C. Hydrolysis was carried outwithout stirring in the presence of 0.5 mg/ml bovine serum albumin (BSA,Sigma, St. Louis, Mo., USA). Reducing sugars were determined usingp-hydroxybenzoic acid hydrazide (PHBAH) assay described in Example 26.

TABLE 1 Relative activity of endoglucanases on carboxymethylcellulose (5mg/ml) at pH 5.0 and 50° C. Endoglucanase Activity on CMC, %Myceliophthora thermophila Cel5A  28 basidiomycete CBS 494.95 Cel5B  44basidiomycete CBS 495.95 Cel5A 100

Example 28 Thermal Stability of Endoglucanases at 40° C.-80° C.

The thermal stability of the purified endoglucanases from Myceliophthorathermophila CBS 117.65, basidiomycete CBS 494.95, and basidiomycete CBS495.95 was determined by incubating enzyme solutions at fivetemperatures (40° C., 50° C., 60° C., 70° C., and 80° C.), and measuringthe residual activity of enzymes on carboxymethylcellulose (CMC).

The enzymes were diluted in 50 mM sodium acetate pH 5.0, which contained3.0 mg/ml BSA, and incubated for 3 hours in 1.1-ml ImmunoWare Microtubesarranged in an 8×12 microplate format (Pierce, Rockford, Ill., USA). BSAwas added in order to prevent possible enzyme adsorption onto theplastic walls of microtubes. The protein concentration in the incubationmixtures was chosen so that each enzyme would give less than 1%conversion of CMC in subsequent assay for CMCase activity.

After a 3 hour incubation, 15 μl aliquots were removed using an8-channel pipettor, and added to 75 μl of CMC solution (6 mg/ml in 50 mMsodium acetate pH 5.0) in a 96-well conical-bottomed microplate (Costar,clear polycarbonate, Corning Inc., Acton, Mass., USA). The residualCMCase activity was then measured as described in Example 27, andexpressed as a percentage of the initial CMCase activity (Table 2).

At 40° C. and 50° C., all three endoglucanases were stable and retained98-100% of the initial CMCase activity after 3 hours of incubation. At60° C. and 70° C., the Myceliophthora thermophila Cel5A showed betterstability than the two other endoglucanases, and retained 100% and 49.3%of the initial CMCase activity after a 3-hour incubation, respectively.None of the endoglucanases were stable at 80° C.

TABLE 2 Residual CMCase activity of endoglucanases after incubation forthree hours at pH 5.0 and 40-80° C. Residual CMC-ase activity, % ofinitial activity Endoglucanase 40° C. 50° C. 60° C. 70° C. 80° C.Myceliophthora thermophila 100 100 100 49.3 3.8 Cel5A basidiomycete CBS494.95 100 98 28.0 5.0 4.1 Cel5B basidiomycete CBS 495.95 100 99 6.4 2.40.8 Cel5A

Example 29 Relative Activity of Endoglucanases on PhosphoricAcid-Swollen Cellulose at 40-70° C.

The activity of the purified endoglucanases from basidiomycete CBS494.95 and basidiomycete CBS 495.95 on phosphoric acid-swollen cellulose(PASC) was determined by measuring the concentration of the reducingsugars (RS) released during initial hydrolysis of PASC (2 mg/ml) in 50mM sodium acetate pH 5.0. Hydrolysis was carried out without stirring inthe presence of 0.5 mg/ml bovine serum albumin (BSA, Sigma, St. Louis,Mo., USA). The enzymes were diluted so that the RS concentration wouldincrease linearly during the initial 30 to 90 minutes of hydrolysis, andthe degree of PASC conversion would not exceed 2% during this time.Reducing sugars were determined using the p-hydroxybenzoic acidhydrazide (PHBAH) assay as described in Example 26.

The relative activity as a function of temperature of the endoglucanasesfrom basidiomycete CBS 494.95 and basidiomycete CBS 495.95 is shown inFIG. 16. The activity is shown as percentage of the activity of theendoglucanase from basidiomycete CBS 495.95 at 60° C. For bothendoglucanases, the activity on PASC attained the maximum value atT_(opt.)=60° C.

Example 30 Long-Term Hydrolysis of Phosphoric Acid-Swollen Cellulose at40-70° C.

Hydrolysis of phosphoric acid-swollen cellulose (PASC, 2 mg/ml) by thepurified endoglucanases from basidiomycete CBS 494.95 and basidiomyceteCBS 495.95 was carried out for 45 hours in 50 mM sodium acetate pH 5.0containing 0.5 mg/ml BSA at four temperatures, 40° C., 50° C., 60° C.,and 70° C. The endoglucanases were used at three protein loadings,0.056, 0.167, or 0.5 mg per g of PASC. The reactions with the initialvolume of 1 ml were run without stirring in 1.1-ml ImmunoWare Microtubesarranged in an 8×12 microplate format (Pierce, Rockford, Ill., USA).

One hundred microliter aliquots were removed from the reactions atdifferent time points (1, 1.5, 3, 6, 21, 27, and 45 hours) using an8-channel pipettor, and added to 25 μl of 2% NaOH in MultiScreen HV96-well filtration plate (Millipore, Bedford, Mass., USA). The collectedsamples were vacuum-filtered into a flat-bottomed microplate to removethe PASC residue. The filtrates were analyzed for reducing sugars by thep-hydroxybenzoic acid hydrazide (PHBAH) assay as described in Example26.

FIG. 17 shows the relative conversion of PASC after a 45 hour incubationwith the endoglucanases from basidiomycete CBS 494.95 and basidiomyceteCBS 495.95 (0.5 mg protein per g of PASC) as a function of temperature.The relative conversion is shown as a percentage of the conversionobtained after 45-hour incubation with basidiomycete CBS 495.95 at 50°C. Temperature profiles obtained at two other protein loadings, 0.056and 0.167 mg protein per g of PASC, had similar shapes. For bothendoglucanases, the optimal temperature for long-term hydrolysis of PASCwas 50° C.

Example 31 Characterization of Endoglucanases on Various PolysaccharideSubstrates at 50° C.

The purified endoglucanases from Myceliophthora thermophila CBS 117.65,basidiomycete CBS 494.95, and basidiomycete CBS 495.95 were evaluated inthe hydrolysis of various polysaccharides at pH 5.0 (50 mM sodiumacetate buffer) and 50° C. The results were compared with those forrecombinant Trichoderma reesei Cel7B (EGI) endoglucanase. RecombinantTrichoderma reesei Cel7B (EGI) endoglucanase produced by Aspergillusoryzae can be prepared according to Takashima et al., 1998, Journal ofBacteriology 65: 163-171.

The polysaccharides included pretreated corn stover (PCS), phosphoricacid-swollen cellulose (PASC), carboxymethylcellulose (CMC), bacterialcellulose (BC), Avicel, xylan, xyloglucan, arabinoxylan, mannan andgalactomannan. All substrates were used at 5 mg/ml, with the exceptionof bacterial cellulose, which was used at 0.9 mg/ml.

Reactions with an initial volume of 1 ml were carried out for 24 hourswith intermittent stirring in Eppendorf 96 DeepWell Plates (1.2 ml, VWRScientific, West Chester, Pa., USA) capped with Eppendorf 96 DeepWellMats (VWR Scientific, West Chester, Pa., USA). Unless otherwisespecified, the enzymes were loaded at 5 mg of protein per g of solids.

After 24 hours, 20 μl aliquots were removed from the hydrolysisreactions using an 8-channel pipettor, and added to 180 μl of 102 mMNa₂CO₃-58 mM NaHCO₃) in a MultiScreen HV 96-well filtration plate(Millipore, Bedford, Mass., USA) to terminate the hydrolysis. Thesamples were vacuum-filtered into a flat-bottomed microplate. Afterappropriate dilution, the filtrates were analyzed for reducing sugarsusing the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described inExample 26.

Table 3 shows relative conversion of various polysaccharides by theendoglucanases after 24-hour incubation. The relative conversion wascalculated as a percentage of conversion obtained after 24-hourhydrolysis of 1,4-β-D-mannan by basidiomycete CBS 495.95 Cel5Aendoglucanase. Endoglucanases from glycoside hydrolase (GH) family 5 hadrelatively high activity on mannan and galactomannan, but low activityon xylan, xyloglucan and arabinoxylan. In contrast, Trichoderma reeseiCel7B had relatively high activity on xylan, xyloglucan andarabinoxylan, but low activity on mannan and galactomannan. The GH5endoglucanases showed better hydrolysis of PASC (insoluble unsubstitutedamorphous cellulose) than CMC (soluble substituted cellulosederivative). The GH5 endoglucanases had low activity on insolublesubstrates with high degree of crystallinity: bacterial cellulose,Avicel, and PCS.

TABLE 3 Relative conversion of various polysaccharide substrates (5mg/ml) by endoglucanases (5 mg protein per g solids); pH 5.0, 50° C., 24hours Myceliophthora basidiomycete basidiomycete Trichoderma thermophilaCBS 494.95 CBS 495.95 reesei Substrate Cel5A Cel5B Cel5A Cel7BPretreated corn stover 2 6 6 10 (PCS) Phosphoric acid 10 40 70 38swollen cellulose (PASC)** Carboxymethylcellulose 12 12 12 14 (CMC)**Bacterial cellulose 1 5 5 5 (BC)* Avicel (microcrystalline 1 2 3 5cellulose) Birchwood xylan 0 3 2 51 Tamarind xyloglucan 0 1 1 87 Wheatarabinoxylan 6 7 8 81 1,4-β-D-Mannan 70 73 100 2 Carob galactomannan 5254 62 3 *Initial concentration of bacterial cellulose was 0.9 mg/ml**All endoglucanases were used at 0.25 mg protein per g solids forhydrolysis of PASC and CMC

Example 32 Hydrolysis of Soluble Beta-Glucan from Barley byEndoglucanases at 60° C.

The activity of the endoglucanases from Myceliophthora thermophila CBS117.65, basidiomycete CBS 494.95, and basidiomycete CBS 495.95 onsoluble beta-glucan from barley (medium viscosity, 230 kDa, MegazymeInternational Ireland Ltd., Bray, Ireland) was determined at pH 5.5 (50mM sodium acetate with 0.02% sodium azide) and 60° C. The results werecompared with those for Trichoderma reesei Cel7B (EGI) endoglucanase.Recombinant Trichoderma reesei Cel7B (EGI) endoglucanase can be preparedas described in Example 31.

The initial concentration of beta-glucan in the hydrolysis reactions was1.0% (w/v). One ml reactions were run without stirring in Eppendorf 96DeepWell Plates (1.2 ml, VWR Scientific, West Chester, Pa., USA). Theenzymes were used at three protein loadings, 0.05, 0.1, and 0.2 mg per gof glucan. In a buffer control, the endoglucanases were substituted with50 mM sodium acetate pH 5.5 containing 0.02% sodium azide.

Aliquots were removed from the hydrolysis reactions at 2 hours and 24hours, diluted with deionized water, and analyzed for reducing sugarsusing the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described inExample 26. The relative conversion of beta-glucan as a function ofprotein loading at two incubation times, 2 hours and 24 hours, is shownin FIGS. 18 and 19, respectively. The relative conversion is shown as apercentage of conversion obtained after 24 hour hydrolysis ofbeta-glucan by Myceliophthora thermophila CBS 117.65 Cel5A endoglucanase(0.2 mg protein per g of glucan).

The endoglucanases from basidiomycete CBS 494.95 and basidiomycete CBS495.95 showed similar performance in hydrolyzing beta-glucan, andproduced no additional increase in reducing sugar concentration after 2hour hydrolysis. In contrast, the endoglucanases from Myceliophthorathermophila and Trichoderma reesei continued to produce new reducingend-groups beyond the 2 hour incubation time. The Myceliophthorathermophila endoglucanase showed better performance in hydrolyzingbeta-glucan than the basidiomycete CBS 494.95 Cel5B endoglucanase andbasidiomycete CBS 495.95 Cel5A endoglucanase.

Deposit of Biological Material

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:

Deposit Accession Number Date of Deposit E. coli strain PBCel5C NRRLB-30900N Feb. 23, 2006 E. coli strain pClC161 NRRL B-30902 Feb. 23, 2006E. coli strain pClC453 NRRL B-30903 Feb. 23, 2006 E. coli strain pClC486NRRL B-30904 Feb. 23, 2006The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polypeptide having endoglucanase activity, selected fromthe group consisting of: (a) a polypeptide comprising an amino acidsequence which has at least 90% sequence identity with the maturepolypeptide of SEQ ID NO: 6; (b) a polypeptide which is encoded by apolynucleotide which hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:5, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 5, or (iii) a full-length complementary strand of(i) or (ii); and (c) a polypeptide which is encoded by a polynucleotidehaving at least 90% sequence identity with the mature polypeptide codingsequence of SEQ ID NO:
 5. 2. The polypeptide of claim 1, which comprisesor consists of the amino acid sequence of SEQ ID NO:
 6. 3. Thepolypeptide of claim 1, which is encoded by a polynucleotide comprisingor consisting of SEQ ID NO:
 5. 4. The polypeptide of claim 1, which isencoded by the polynucleotide contained in plasmid pCIC453 which iscontained in E. coli NRRL B-30903.
 5. An isolated polynucleotidecomprising a nucleotide sequence which encodes the polypeptide ofclaim
 1. 6. The isolated polynucleotide of claim 5, comprising at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 5in which the mutant nucleotide sequence encodes the mature polypeptideof SEQ ID NO:
 6. 7. A nucleic acid construct comprising thepolynucleotide of claim 5 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.
 8. A recombinant host cell comprising the nucleic acid constructof claim
 7. 9. A method for producing the polypeptide of claim 1comprising: (a) cultivating a cell, which in its wild-type form iscapable of producing the polypeptide, under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 10. Amethod for producing the polypeptide of claim 1 comprising: (a)cultivating a host cell comprising a nucleic acid construct comprising anucleotide sequence encoding the polypeptide under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.11. A method for producing a mutant of a parent cell, which comprisesdisrupting or deleting a nucleotide sequence encoding the polypeptide ofclaim 1, which results in the mutant producing less of the polypeptidethan the parent cell.
 12. A method for producing the polypeptide ofclaim 1, comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding a polypeptide having endoglucanaseactivity under conditions conducive for production of the polypeptide;and (b) recovering the polypeptide.
 13. A transgenic plant, plant partor plant cell, which has been transformed with a polynucleotide encodingthe polypeptide of claim
 1. 14. A method of degrading or converting acellulosic material, comprising: treating the cellulosic material with acomposition comprising an effective amount of a polypeptide havingendoglucanase activity of claim
 1. 15. A method of degrading orconverting a cellulosic material, comprising: treating the biomass witha composition comprising the host cell of claim
 8. 16. A method ofproducing a substance, comprising: (a) saccharifying a cellulosicmaterial with a composition comprising an effective amount of apolypeptide having endoglucanase activity of claim 1, (b) fermenting thesaccharified cellulosic material of step (a) with one or morefermentating microorganisms; and (c) recovering the substance from thefermentation.
 17. The polypeptide of claim 1, comprising or consistingof the amino acid sequence of the mature polypeptide of SEQ ID NO: 6.18. The polypeptide of claim 17, wherein the mature polypeptide is aminoacids 16 to 397 of SEQ ID NO:
 6. 19. The polypeptide of claim 1, whichis encoded by a polynucleotide comprising or consisting of the maturepolypeptide coding sequence of SEQ ID NO:
 5. 20. The polypeptide ofclaim 19, wherein the mature polypeptide coding sequence is nucleotides84 to 1229 of SEQ ID NO:
 5. 21. The polypeptide of claim 1, comprisingan amino acid sequence which has at least 95% sequence identity with themature polypeptide of SEQ ID NO:
 6. 22. The polypeptide of claim 1,comprising an amino acid sequence which has at least 97% sequenceidentity with the mature polypeptide of SEQ ID NO:
 6. 23. Thepolypeptide of claim 1, comprising an amino acid sequence which has atleast 98% sequence identity with the mature polypeptide of SEQ ID NO: 6.24. The polypeptide of claim 1, comprising an amino acid sequence whichhas at least 99% sequence identity with the mature polypeptide of SEQ IDNO:
 6. 25. The polypeptide of claim 1, which is encoded by apolynucleotide having at least 95% sequence identity with the maturepolypeptide coding sequence of SEQ ID NO:
 5. 26. The polypeptide ofclaim 1, which is encoded by a polynucleotide having at least 97%sequence identity with the mature polypeptide coding sequence of SEQ IDNO:
 5. 27. The polypeptide of claim 1, which is encoded by apolynucleotide having at least 98% sequence identity with the maturepolypeptide coding sequence of SEQ ID NO:
 5. 28. The polypeptide ofclaim 1, which is encoded by a polynucleotide having at least 99%sequence identity with the mature polypeptide coding sequence of SEQ IDNO:
 5. 29. The polypeptide of claim 1, which is encoded by apolynucleotide which hybridizes under at least very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:5, (ii) the genomic DNA sequence of the mature polypeptide codingsequence of SEQ ID NO: 5, or (iii) a full-length complementary strand of(i) or (ii).